NZ715706A - 3-aryl propiolonitrile compounds for thiol labeling - Google Patents

3-aryl propiolonitrile compounds for thiol labeling Download PDF

Info

Publication number
NZ715706A
NZ715706A NZ715706A NZ71570614A NZ715706A NZ 715706 A NZ715706 A NZ 715706A NZ 715706 A NZ715706 A NZ 715706A NZ 71570614 A NZ71570614 A NZ 71570614A NZ 715706 A NZ715706 A NZ 715706A
Authority
NZ
New Zealand
Prior art keywords
mmol
nmr
group
mhz
compound
Prior art date
Application number
NZ715706A
Other versions
NZ715706B2 (en
Inventor
Alain Wagner
Oleksandr Koniev
Original Assignee
Univ Strasbourg
Centre Nat Rech Scient
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Strasbourg, Centre Nat Rech Scient filed Critical Univ Strasbourg
Publication of NZ715706A publication Critical patent/NZ715706A/en
Publication of NZ715706B2 publication Critical patent/NZ715706B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/34—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring with cyano groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by unsaturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/35—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/37—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by etherified hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/41—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by carboxyl groups, other than cyano groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
    • C07C255/43—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms the carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
    • C07C255/44—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms at least one of the singly-bound nitrogen atoms being acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
    • C07C271/28—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a non-condensed six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/42—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C07C309/42—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having the sulfo groups bound to carbon atoms of non-condensed six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/02—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of thiols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/42—Y being a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
    • C07C331/16—Isothiocyanates
    • C07C331/28—Isothiocyanates having isothiocyanate groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/22—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
    • C07D265/38—[b, e]-condensed with two six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D311/88—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D337/00—Heterocyclic compounds containing rings of more than six members having one sulfur atom as the only ring hetero atom
    • C07D337/02—Seven-membered rings
    • C07D337/04—Seven-membered rings not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/04—Esters of boric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/0803—Compounds with Si-C or Si-Si linkages
    • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/54—Quaternary phosphonium compounds
    • C07F9/5442—Aromatic phosphonium compounds (P-C aromatic linkage)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Oncology (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
  • Pyrrole Compounds (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to a process for labeling compounds comprising thiol moieties with 3-arylpropiolonitrile compounds, to 3-arylpropiolonitrile compounds substituted with tag moieties and to specific 3-arylpropiolonitrile linkers.

Description

-aryl propiolonitrile compounds for thiol labeling The present invention relates to a s for labeling compounds comprising thiol moieties with 3—ary1propiolonitrile compounds, to propiolonitrile compounds substituted with tag moieties and to specific 3-arylpropiolonitrile linkers.
Background of the invention Over 90% of the human proteins contain cysteines, while in silico digest of the human proteome revealed that only about 15% of all human tryptic peptides detectable by mass spectroscopy (MS) contain at least one ne in their sequence. This observation combined with the presence of a highly reactive thiol group on its side chain makes cysteine an attractive target for chemical labeling. Cysteine is the only coded amino acid that carries a nucleophilic sulfhydryl (or thiol) group (-SH), which largely exceeds the reactivity of any other nucleophilic function susceptible to be present in proteins. As a result, chemospecific ne derivatization is by far the most widely used method for chemical tagging of proteins. Among the vast number of chemical ne modification methods ed in literature so far, reagents such as N—substituted maleimides, 4-vinylpyridines and iodoacetamides are most commonly used. All of them possess drawbacks preventing them from being ideal methodology for cysteine labeling, though being suited for this task. These drawbacks are mainly presence of undesired side reactions, in ular for iodoacetamides and maleimides, and instability of addition product in biological nments due to reversible thiol exchange and other side ons.
The present invention relates to a process for labeling compounds sing at least one thiol moiety, such as cysteine, with compounds comprising a tag moiety and a 3- arylpropiolonitrile moiety. Said compounds and their addition products with ne tives show an unexpected stability in a wide range of conditions. The process for labeling compounds comprising thiol moieties of the invention can thus be used for a wide range of applications.
WO 20151001117 2 Summam of the invention The first object of the invention is a process for the preparation of a ed compound sing a thiol moiety, comprising contacting a compound comprising a thiol moiety with a compound of formula (I) wherein R1 to R5 are as described below, and wherein at least one of R1 to R5 comprises a tag moiety.
Another object of the invention is a compound of a (I) wherein R1 to R5 are as defined below, and wherein at least one of R1 to R5 comprises a tag moiety.
Another object of the invention is a compound of formula (II) wherein R1 to R5 are as defined below, and wherein at least one of R1 to R5 is different from a hydrogen atom.
Another object of the invention is a compound of formula (111) R2 R1 S—Re wherein R6-S corresponds to the moiety of the compound comprising at least one thiol moiety as identified above.
WO 20151001117 3 Brief description of the figures Fig l: Evolution of ity of fluorescence over time obtained in human plasma for the same concentration of compounds A and B.
Fig 2: a) Evolution of intensity of fluorescence over time obtained in cellula for the same concentration of compounds A and B. b) Intensity ratio compound B/compound A at different times in cellulo. c) Microscope pictures of cells treated with compounds A and B, superimposition of TAMRA and Hoechst labeling. White spots around nucleus correspond to hydrolyzed probe. A — arylpropiolonitrile probe, B — maleimide probe.
Fig 3: HPLC monitoring of the hydrolysis of phenylmaleimide (2) in PBS (lx, pH 7.6) at lmM tration at 25°C; the peaks correspond, from the earliest to the O \ (3%” O WH N 0 O , COOH, and 2O latest, to ; sion was monitored by disappearance of the ng material (a). Pseudo first order rate nt for the reaction was determined by plotting the ln([phenylmaleimide]) versus time and analyzing by linear regression. The constant corresponds to the absolute value of ined slope (b).
Fig 4: HPLC monitoring of hydrolytic stability of N—(4-(cyanoethynyl)phenyl)acetamide (pNHAc-APN, 11) in PBS (lx, pH 7.6) at lmM concentration at 25°C. Conversion was red by disappearance of the starting material; the peaks correspond, from the earliest to the latest, to AcN : :N and 11 .
. No detectable convers1on of arylpropiolonitrile 11 was observed after 5 hours of monitoring.
Fig 5: MTT test results for compounds 1, 2, 3, 5, 7, 11, 10 and 9.
Fig 6: a) schematic structure and measured DLS spectra of CD38 and CD38 A275C mutant; b) scheme of CD38 A275C modification with 49; c) DLS ic Light Scattering) spectrum of the resulting conjugate.
WO 01117 4 Fig 7: Gel electrophoresis of CD38-C375 mutant labeled with a compound according to the invention and with the corresponding maleimide compound, before purification (a) and after ation (b).
Fig 8: Strategy of preparation of the antibody-TAMRA conjugate with a compound according to the invention, and the comparison with the corresponding maleimide.
Fig 9: Gel electrophoresis of the conjugates obtained respectively with the compound according to the invention (CBTF) and with the corresponding maleimide (SMCC).
Fig lO:Mass spectrum for the conjugate obtained with the compound according to the invention.
Fig ll:Zoom on the mass spectrum of figure 10.
Fig 12:Mass spectrum obtained with the maleimide.
Fig l3:Zoom on the mass spectrum of figure 12.
Fig eral scheme of direct conjugation of the compound 58 to lly reduced Trastuzumab.
Fig 15:SDS-PAGE analysis of the obtained conjugates shows that nd 58 is covalently attached to the antibody Fig 16: General scheme of ging of antibody fragments using compounds 33 and 34.
Fig l7:SDS-PAGE analysis shows that antibody nts are successfully bridged by compounds 33 and 34.
WO 20151001117 5 Detailed description of the invention The first object of the invention is a process for the ng of a compound comprising a thiol moiety, comprising contacting said compound comprising a thiol moiety with a compound of a (1) R4 wherein each of R1 to R5 is independently selected in the group consisting of: - hydrogen atoms, - alkyl, alkene or alkyne , optionally interrupted by at least one heteroatom selected among 0, N and S, 1 0 - aryl groups, - alkoxy groups, - halogen atoms, - amino (-NRR’) groups, wherein R and R’ are independently hydrogen atoms, alkyl, alkene, alkyne or aryl groups as defined below, - hydroxylamine (-ONH2) group, - hydrazine (-NH-NH2) group, - nitro (—N02) group, - azido (-N3) group, - diazoniurn (-N2+) group, optionally in presence of a counterion, - maleimide group, - alkyl- or aryl-carboxyl (-C(=O)OR) groups, wherein R is as bed above, - alkyl- or aryl-carbonyl (-C(=O)R) groups, wherein R is as bed above, - hydroxyl (-OH) group, - boronic acid -B(OR’ ’)2 group, wherein R’ ’ is a hydrogen atom or an alkyl group, - phosphine or onium groups, - isocyanate (-N=C=O) or ocyanate (-N=C=S) group, - chlorosulfonyl (-SOzCl) group, - a -O-C(=O)-C(N2)-CF3 group or a -C(=O)—C(N2)_CF3 group, - activated esters, such as -C(=O)-NHS, wherein NHS stands for N- hydrosuccinimidyl, perfluorinated esters, and acylureas, WO 20151001117 6 - a -CEC-CEN group, - tags, and - alkyl groups substituted by at least one of the previously listed groups, wherein at least one of R1 to R5 comprises, preferably is, a tag moiety.
Two of R1 to R5 may alternatively form together and with the carbon atoms of the phenyl ring to which they are linked a mono or polycyclic ring, saturated, unsaturated or aromatic, optionally comprising at least one heteroatom such as P, O or S.
The tag moiety that is sed in the compound of formula (I) may be directly bonded to the phenyl ring. It may also be bonded to the phenyl ring h a “linker” group, such as a C00, a NH-C(=O)—NH, a NH-C(=O)—O, a triazole, or a CONH group.
It may also be present as a tuent of one of the R1 to R5 groups as described above.
In a preferred embodiment, R2, comprises, preferably R3 is, a tag moiety.
In the present invention, the term “alkyl” relates to a linear, cyclic or branched hydrocarbon group comprising from 1 to 20 carbon atoms, preferably from 1 to 6 carbon atoms, in particular from 1 to 3 carbon atoms. Among alkyl groups can be cited for instance the methyl, ethyl, n-propyl, pyl, n-butyl, sec—butyl, tert-butyl, n- pentyl, n-hexyl and cyclohexyl groups. An alkyl group according to the invention may be interrupted by at least one heteroatom selected from Si, N, O and S. Among alkyl groups interrupted by at least one heteroatom may be cited the polyethyleneglycol groups of formula -CH2)n-OH, wherein n is from 1 to 1000, preferably from 1 to 100, in particular from 1 to 8. An alkyl group according to the invention may be tuted by at least one halogen atom.
The term “alkene” relates to an alkyl group as defined above, further comprising at least one C=C double bond.
The term “alkyne” relates to an alkyl group as defined above, further comprising at least one CEC triple bond. Among alkyne groups can be cited for instance acetylene and cyclooctyne groups.
The term “alkoxy” s to an alkyl group as defined above linked to the rest of the molecule via an oxygen atom.
WO 20151001117 7 The term “aryl” relates to a group comprising at least one planar ring comprising a conjugated 7: system made of double bonds and/or non-bonding doublets, wherein each atom of the ring comprises a p orbital, the p orbitals overlay each other, and the delocalization of the n electrons lowers the le energy. Preferably, the aryl group is a hydrocarbon aryl group, optionally comprising at least one atom selected from N, O and S. Preferably, an aryl group is selected from the group consisting of , pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, thiophenyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, dihydroisoxasolyl, triazolyl, diazinyl, inyl, pyrazolyl and naphthyl groups. In particular, an aryl group is selected from the group consisting of isoxazolyl, oisoxasolyl, triazolyl, diazinyl, tetrazinyl and pyrazolyl groups.
The term “halogen” relates to an atom selected from the group consisting of F, Cl, Br and I atoms. Preferably, a halogen is a C1 or Br atom.
The optical and geometrical isomers, racemates, tautomers, salts, hydrates, solvates and mixtures thereof of the compounds are also encompassed by the scope of as (I), (II), (III) and (IV) of the t invention.
When the compounds according to the invention are in the forms of salts, they are preferably pharmaceutically acceptable salts. Such salts include ceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
Acid addition salts include salts of inorganic acids as well as organic acids.
Representative examples of suitable inorganic acids e hydrochloric, hydrobromic, odic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable c acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fiamaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruVic, lic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, ic, c, palmitic, EDTA, glycolic, p—aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, utarates and the like. Further WO 20151001117 8 examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2.
Preferably, the salt does not comprise any thiol moiety.
The “counterion” can be any ion appropriate for compensating the charge of the diazonium group, and may be easily chosen by anyone of ordinary skill in the art. For instance, the counterion may be selected from the group consisting of halogenates, BF4', N03", HSO4‘, PF6' CH3COO', N(SOzCF3)2', CngOs', CH3803', ', (CH30)(H)P02' and N(CN)2'.
In the present invention, the hydroxyl (OH), amino (NH2 or NHR) and carboxyl (COOH) groups may be ted with appropriate protecting groups. One can refer to T. W. Green, P. G. M. Wuts, Protective Groups in Organic sis, Wiley- Interscience, New York, 1999.
Among protecting groups for yl groups may be cited acetyl (Ac), benzoyl (Bz), benzyl (Bn), B-Methoxyethoxymethyl ether (MEM), Dimethoxflrityl, [bis-(4- methoxyphenyl)phenylmethyl] (DMT), Methoxymethyl ether (MOM), Methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT), p-Methoxybenzyl ether (PMB), Methylthiomethyl ether, yl (PiV), Tetrahydropyganyl (THP), Tetrahydrofuran (THF), andm (triphenylmethyl, Tr).
Among protecting groups for amino groups may be cited t-butyl carbamate (Boc), 2- trimethylsilylethyl carbamate (Teoc), l—(l-Adamantyl)—l-methylethyl carbamate (Adpoc), l-Methyl-l-(4-biphenyl)ethyl carbamate (Bpoc), l-(3,5—Di-t—butylphenyl)-lmethylethyl ate (t—Bumeoc), antyl Carbamate (Adoc), p-Methoxybenzyl carbamate (Moz), 9-Anthrylmethyl carbamate, Diphenylmethyl Carbamate, 9- Fluorenylmethyl carbamate (Fmoc), 9-(2-Sulfo) Fluoroenylmethyl carbamate, 9-(2,7- dibromo)Fluorenylmethyl carbamate, 2,7-Di-t-butyl-[9-(lO, lO-dioxo-lO,lO, 10,10- tetrahydrothioxanthyl)]methylcarbamate (DBD—Tmoc), 2-(N,N— ohexylcarboxamido)ethlearbamate, 2-Phosphonioethyl carbamate , 2- Phenylethyl carbamate, Benzyl carbamate (Cbz), Allyl ate (Alloc), l- Isopropylallyl carbamate ), 4-Nitrocinnamyl Carbamate (Noc), 8-Quinolyl carbamate and N—Phtalimide.
WO 20151001117 9 Among protecting groups for carboxyl groups may be cited methyl esters, benzyl esters, tert-butyl esters, silyl esters, 2,6-dimethylphenol, 2,6-diisopropylphenol, and 2,6-di-tert- butylphenol.
The term “tag” or “tag moiety” relates to a chemical group riate for allowing one or several of the following: — detection of the compound, - vectorization of an agent of interest by the nd, - solubilization of the compound, - stabilization of the compound, - improvement of the extraction and/or purification of the compound, - modification of at least one of the ADME (Administration Distribution Metabolisation Excretion) parameters of the compound; - addition of bioactivity to the compound; - addition of an appropriate functionality for click-chemistry.
The compounds comprising such tags and according to the invention can therefore be used as a tool for detection, vectorization of an agent of interest, solubilization, stabilization, improvement of the extraction and/or purification, modification of at least one of the ADME (Administration Distribution Metabolisation Excretion) parameters; addition of bioactivity; and/or addition of an appropriate fiinctionality for click- chemistry.
A chemical group appropriate for allowing detection of the compound of the invention may be any chemical group that can be identified and/or fied by any que of analysis known in the art. Among tags for detection can be cited fluorescent, such as fluorescent probes, such as cein, quantum dots, cyanine dyes Cy3® and Cy5®, Alexa Fluor® dyes, Dylight fluor® dyes, IRIS® Dyes, Seta® dyes, SeTau® dyes, SRfluor® dyes, Square® dyes, Nile red, iFLi[AMIJ or carboxytetramethylrhodamine (TAMRA); r ic Resonance (NMR) tags, such as xenon or nides (in particular m Tb or europium Eu); magnetic resonance imaging (MRI) contrast agents such as Gd es; mass spectrometry tags such as tris(2,4,6- WO 20151001117 10 trimethoxyphenyl)phosphonium (TMPP) or isotope-coded tags; infrared (IR) tags; positron emission tomography (PET) tags; single-photon emission computed tomography (SPECT) tags; tritium or deuterium atoms; microscopy tags such as gold nanoparticles; quenchers such as dabsyl (dimethylaminoazobenzene ic acid).
A chemical group appropriate for allowing vectorization of an agent of interest with the compound of the invention may be any chemical group or biological moiety appropriate for helping the compound and/or the agent of interest to reach the appropriate tissue or organ, such as liver or bladder. For instance, the vectorization tag may be a al group able to form micelles, reverse-micelles or liposomes, such as an amphiphilic chemical group, a nano or microparticle, a viral vector, a r, a folate, an ammonium group, a peptide, an EGFR (Epidermal Growth Factor Receptor) ligand or an antibody.
A chemical group appropriate for allowing stabilization of the compound is a chemical group that affords sing the half—life of the compound, preferably in viva. For instance, the stabilization tag may be albumin, such as Human Serum Albumin HSA or Bovine Serum Albumin BSA.
A chemical group appropriate for allowing modification of the ADME parameters of the compound can be for instance a therapeutic agent, a drug, a prodrug, a polyethylene glycol of a -(OCH2-CH2)n-OR”, wherein R” is a hydrogen atom or an alkyl group, wherein n is from 1 to 1000, preferably from 1 to 100, in particular from 1 to 8, a peptide, such as e-Alanine-Serine (Pro-Ala-Ser) or poly-Glu, a polypeptide, such as XTEN recombinant polypeptide, a lipid, such as palmitic acid, a carbohydrate, yethyl starch, a nucleic acid, such as DNA or RNA, in particular siRNA. The term “prodrug” relates to a variant of a drug that can be transformed in viva into a drug.
The term de” relates to a peptide comprising from 1 to 20, preferably from 1 to , cids.
The selection of the riate tag, such as the determination of the number of ethylene glycol es, can be easily adjusted by one of ordinary skill in the art depending of the desired ADME modification.
WO 20151001117 11 A chemical group appropriate for allowing extraction and/or purification of the compound may be any chemical group that favors and/or facilitates the extraction and/or ation of the compound of the invention. Among extraction and/or purification-tags can be cited biotin, chelating tags such as DTPA (diethylenetriaminepentaacetic acid), EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid), NTA (nitrilotriacetic acid) and D4 (octamethylcyclotetrasiloxane), protein tags such as ginine or polyhistidine tags, preferably His6 or Hile tags, ag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag, -binding , glutathione S-transferase tag, maltose-binding protein, NusA, TrxA and DsbA tags, boronic tags such as [(3-oxospiro[isobenzofiiran-1(3H),9'- [9H]xanthene]-3 ’,6’-diyl)bis(iminomethylene—2, l -phenylene)]bis-(9Cl), perfluoroalkyl groups, ionic (cationic or anionic) groups, such as ammonium groups, and solid surfaces such as ric materials, in particular polyethylene beads, nanoparticles, in particular magnetic nanoparticles, chips, silica beads or silica wafers.
A chemical group appropriate for addition of bioactivity may be for instance a chemical group comprising at least one sotope, such as 131I, 90Y, 89Sr, or 153Sm, or a derivative thereof.
A chemical group appropriate for reacting in click-chemistry may be for instance a chemical group selected from the group ting of azides (such as N3) and strained alkynes, in particular cyclic alkynes. Among cyclic alkynes may be cited for instance the bicyclononyne (BCN) and tetramethylthiepinium (TMTI) moieties.
In an embodiment, each of R1 to R5 is independently ed in the group consisting of: - alkyne groups, - amino groups, - hydroxylamine (-ONH2) groups, - hydrazine (-NH-NH2) groups, - azido (N3) groups, - diazonium (Nf) groups, preferably in presence of a counterion, - maleimide groups, wo 2015;001117 12 2014/064387 - carboxylic acid , - aldehyde (-CHO) groups, - boronic -B(OR’ ’)2 groups, wherein R” is as described above, and - activated esters.
In a preferred ment, none of R1 to R5 comprises a free SH group. In a preferred embodiment, R1 and/or R5 do not comprise any nucleophilic group, such as amino groups, hydroxylamine groups, hydrazine groups or hydroxyl groups.
In specific embodiments, l, 2, 3, 4 or 5 of R1 to R5 are different from hydrogen atoms.
In specific embodiments, at least one of R1 to R5 comprises at least one moiety that is appropriate for further forming a covalent bond with a chemical group selected in the group ting of thiol (SH) moieties, amine (NHz) moieties and carboxylic acid (COOH) es. Among moieties appropriate for forming a covalent bond with thiol moieties, one can cite maleimide es. Among moieties appropriate for forming a covalent bond with amine moieties, one can cite NHS-ester moieties.
Among compounds of formula (I) according to the present invention may be cited the following compounds: N N N N || || || || || || || H H H N‘Boc NH2 NH2 N‘Boc o o o o HO o HO o + + 36 68 69 70 W0 01117 13 HN Q fill El 46: \\ W0 01117 14 TFA‘ \ \0 0’ o 55 / g?:5 0/ /O 0/ HN 0 73 40 INI N Q INI _N+_ I H —~+— II IN H o m) 0 O O O O 1:1F F F FmocHN F F F O OH 76 SO3Na 77 78 18': CF218F 125' 79 so 81 82 WO 20151001117 15 2014/064387 ° and N%©~N\\ /_/_SO3H N N Among compounds of formula (I) may also be cited the following compounds: N 20 W0 01117 O NH o o I I \/\o I/N 0 OH N \\ / N WO 20151001117 17 2014/064387 O OH N: : N\’MONOV\ONO\/\o/\)LNH O OH 0%HN ”NOWONOWONOWONOWNHZ fi0 51 WO 20151001117 18 o\\\ HN 0 N/N:N N: : Wowo~°v\o/\/°j // m , ’N / / 0 N / JL N" N \N/\/\N F I H H F F 53 01‘ 63 sis of comgonnds 0f the invention Compounds of formula (I) or (II) according to the invention can be synthesized for instance in two steps from the corresponding iodoarene, by coupling with propargyl alcohol, for instance Via a shira coupling. The coupling is preferably followed by ion; for instance, the oxidation may be a tandem oxidation performed with Mn02 in the presence of an ammonia solution. Alternatively, the compounds of the invention can be synthesized by cyanation of arylalkynes. Cyanation may be med for instance with CuCN, arylisocyanates, cyanobenzotriazoles or cyanoimidazoles. ng 0t nds comQrising at least one thiol moiety The compounds of formula (I) may be used in a process for labeling compounds comprising at least one thiol SH moiety. ably, the process of the invention comprises contacting at least one compound of formula (I) with a compound comprising at least one thiol SH moiety, or a sample susceptible to comprise such a compound.
Preferably, the sample is a biological sample, in particular an aqueous sample.
WO 20151001117 19 The term “labeling” in the present invention refers to the formation of a covalent bond between the sulfur atom of the thiol moiety and the propiolonitrile moiety of the compounds of formula (I).
The nd comprising a thiol moiety (R6-SH compound) can be for instance a fluorophore, a er, an amino acid, a peptide, a protein, an enzyme, a drug, a prodrug and/or a drug metabolite.
R6 may be any chemical group that is bonded to a thiol (SH) group to form a “compound comprising a thiol ”. R6 preferably comprises carbon, hydrogen, oxygen, nitrogen, phosphorus, and/or r atoms.
In particular, the compound comprising a thiol moiety can be cystein, or a derivative, such as an ester, thereof, or a peptide or a n comprising at least one cystein residue. Alternatively, the compound comprising a thiol moiety may be a surface presenting at least one free SH group.
Labeling of the compounds comprising a thiol moiety with compounds of formula (I) may be used for a great number of applications.
In a first embodiment, labeling of the compounds comprising a thiol moiety with compounds of formula (I) may be used in the ion and/or quantification of the compound comprising the thiol moiety in a sample. The detection means in the present invention identifying the presence or absence of the desired compound(s) in the sample.
The sample can be any sample susceptible to comprise the compound comprising at least one thiol moiety. For instance, the sample may be a biological sample, for ce a biological fluid, such as blood, plasma, serum, saliva, urine, etc., an extract of natural products, a biological tissue, or a part thereof, or a medium comprising cells.
In a second embodiment, labeling of the compounds comprising a thiol moiety with nds of formula (I) may be used for conjugation of the compound sing a thiol moiety with a moiety that improves its physico-chemical properties. For ce, the ated moiety may improve the solubility of the compound comprising a thiol moiety, or improve its synthesis and/or purification.
WO 20151001117 20 2014/064387 In a third embodiment, labeling of the compounds sing a thiol moiety with compounds of formula (I) may be used for njugation of the compound sing a thiol moiety with a compound of interest, such as a drug, a prodrug, a carbohydrate or a protein.
For ce, a compound of formula (1) comprising a compound of interest as tag may allow selective vectorization and/or binding of the compound of interest to the compound comprising at least one thiol .
Another object of the invention is a compound of formula (I) as defined above, including the described specific embodiments.
The invention also discloses a compound of formula (11) R4 R5 wherein each of R1 to R5 is selected independently in the group consisting of: - hydrogen atoms, - alkyl, alkene or alkyne groups, optionally interrupted by at least one heteroatom selected among 0, N and S, - aryl groups, - alkoxy , - halogen atoms, - amino (-NRR’) groups, wherein R and R’ are independently hydrogen atoms or alkyl, alkene, alkyne or aryl groups as defined above, - hydroxylamine (-ONH2) group, - hydrazine (-NH-NH2) group, - nitro (-N02) group, - azido (—N3) group, - diazonium (-N2+) group, preferably in presence of a counterion, - rnaleimide group, WO 20151001117 21 - alkyl- or arboxyl (—C(=O)OR) , - alkyl- or aryl-carbonyl (-C(=O)R) groups, - hydroxyl (-OH) group, - boronic -B(OR’ ’)2 group, wherein R” is a hydrogen atom or an alkyl group, - phosphine or phosphonium groups, - isocyanate (-N=C=O) or isothiocyanate (-N=C=S) group, - chlorosulfonyl l) group, - a O-C(=O)-C(N2)-CF3 group or a -C(=O)-C(N2)_CF3 group, - activated esters, such as -C(=O)-NHS, perfluorinated esters and acylureas, lO - tags, and - alkyl groups substituted by at least one of the previously listed groups.
The compounds of formula (II) are linkers to which at least one tag may be added to form the compounds of a (I) as described above.
Among the compounds of formula (11) according to the present invention may be cited the following compounds: N N | | | | || || Né Ne c\\S CQO 21 84 WO 1117 22 SE [NI l 1 I”I H | | ‘ ‘ 0 NH 0 NH N SO3Na OH 0 N \ o o / 0 O HO U 89 90 41 N INI H N H INI H INI || ” H H " J, a T T NHMe NHAc WO 2015001117 23 | | N I I 'N' I I II II II n | | || M NO; OMe NHAc 93 1 I I II II N I I I I I I COO” NH2 \\ INI 7 1s 34 \N 35 or 3 Among the compounds of formula (11) according to the t invention may also be cited the following compounds: //N //N & ¢ ¢ | F3C O 13 14 15 5L )1 X N N/\/\O/\/O\/\O/\/\N O H H H WO 1117 24 H H O N\/\/O\/\o/\/O\/\/N\n/Oj<O / N N \ / N //N / \ / % é / o O/ /\OJJ\N 0‘ H 0 26 31 32 YNH H 0 HN ‘\\\MNNO%ONN H S 0 o NH I I ‘N:N 42 61 WO 20151001117 25 HN O F38k0.
O b; O s\ ”V 8 IN _ _N O' ll N\N F M NH; 62 64 65 The process for labeling a compound comprising a thiol moiety according to the invention may further comprise, before the step of contacting the compound comprising a thiol moiety With a compound of formula (I), a preliminary step of preparation of the compound of formula (1), comprising contacting a compound of formula (II) with a compound comprising a tag moiety, or a precursor thereof.
The term rsor” relates in the present case to a chemical group that is able to form, after contacting with the compound of formula (II), the tag moiety.
Preferably, at least one of R1 to R5 is ent from a hydrogen atom.
An object of the ion is a compound of formula (11), n at least one of R1 to R5 comprises, preferably is, a maleimide, an azide (N3) group, an alkyne or a NHS-ester moiety.
W0 20151001117 26 2014/064387 The maleimide and NHS-ester moieties respectively allow fiarther linking of the compound to another thiol or an amine group; the N3 group allows further linking of the compound to another alkyne—group and the alkyne group allows further linking of the compound to another N3 group.
In an embodiment, the compound of formula (11) according to the ion is selected from the following compounds: N N N l | l | | | N N || || || II N HN O HN 0 O O l é) || || 71 —SI— N _ | //N+ 29 3O 19 WO 20151001117 27 The invention also s to compounds of formula (I) or (11), wherein at least one of R1 to R5 is further bonded to a compound of st.
A compound of interest may be for instance a molecule, such as a fluorophore, for instance rhodamine, a group of atoms comprising at least one radioactive atom (14C, 3H, or 131I for instance), a group of atoms of known mass (a mass tag), a ligand, a drug, a therapeutic agent, a biomolecule, such as an antibody, a n, such as BSA (bovine serum albumin), a DNA fragment, a nanoobject, such as a nanoparticle (ie an object or a particle of 0.1 to 1000 nm), or a support, such as a polymer.
When the process of labeling according to the invention is performed with such a compound of formula (I) or (11) wherein at least one of R1 to R5 is further bonded to a compound of interest, the process affords the formation of a conjugate between the compound of interest and the compound comprising a thiol moiety.
In an embodiment, the compound of interest is a biomolecule such as a n or an antibody, and the compound comprising a thiol moiety is a fluorophore such as a compound sing a TAMRA moiety and a thiol moiety (TAMRA-SH).
In an embodiment, the compound of interest is a biomolecule such as a protein or an antibody, and the compound comprising a thiol moiety is a drug or a therapeutic agent.
The conjugate obtained by the s of labeling according to the invention is a therapeutic antibody.
Another object of the ion is a compound of formula (111): R2 R1 S_R6 R4 R5 /// (HI) , wherein R6-S corresponds to the moiety of the nd comprising at least one thiol moiety as defined above. In particular, the compound of formula (III) is of formula (IV): WO 20151001117 28 (1V) wherein R7 and Rs are selected in the group consisting of OH, tag, alkyl, O—alkyl, and peptidic es, wherein the alkyl groups may be substituted by at least one tag moiety. A peptidic moiety is a moiety sing at least one aminoacid, when the moiety preents more than one aminoacid (2, 3, 4, 5, ...), the aminoacids are linked between each other by peptidic bonds. Preferably, the double bond of the compound of formula (III) or (IV) is of (Z) configuration. The tag and alkyl groups are as defined above.
Among compounds of a (111) according to the present invention may be cited the following compounds: TFA' 8+ I l \ / The compounds of formula (I) have surprisingly been found to be more stable in aqueous medium than the corresponding compounds wherein the propiolonitrile moiety is replaced with a maleimide moiety, which is classically used for ng thiol moieties. For instance, compound 1 E: was approximately 25% hydrolyzed after 1h in buffer solution (kobs=7x10'5s'1), while no hydrolysis could be detected for WO 20151001117 29 ACN : :N compound 11 . Interestingly, even after a week at room temperature, no trace of yzed product could be ed for compound 11.
In on, the compounds of a (II) according to the invention showed a marked selectivity towards cysteine compared to other amino-acids which do not se free thiol moieties. Comparatively, the chemoselectivity obtained for the corresponding compounds wherein the 3-arylpropiolonitrile moiety is replaced with a maleimide moiety is lower.
Finally, the compounds of formula (III) as described above have shown to be highly more stable in biological conditions than the corresponding compounds wherein the 3- arylpropiolonitrile moiety is replaced with a maleimide moiety. For instance, the T/ CN addition product NHBn between compound %c~ and cysteine H2Nle/NHBn derivative 3 0 was particularly stable in a wide range of conditions, such as physiological conditions. In particular, said on product was stable in a wide range of pH (from 0 to 14), and no exchange product could be observed after one hour of reaction with an excess of phenylthiol and glutathione.
The invention will be illustrated in more detail with nce to the following examples, but it should be understood that the present invention is not deemed to be limited thereto.
WO 20151001117 30 Example 1: Synthesis of compounds of the invention > S thesis of com ounds of formula II A series of compounds of formula (I) or (II) were synthesized and characterized according to the following ures.
General ols: Sonogashira ng Standard reaction protocols: A. To a degased solution of the proper aryl halide (1 eq., 1 mmol) in DMF (5 mL) and DIPEA (10 eq., 10 mmol), premixed PdC12(PPh3)2 (0.03 eq., 30 umol) and CuI (0.06 eq., 60 umol) were added. Obtained reaction mass was degased, stirred for another 5 minutes, followed by the addition of propargyl alcohol (1.2 eq., 1.2 mmol). The reaction mass was stirred for 1-24 hours (monitored by TLC). 1M HCl (50 mL) was added (if contains free amino groups, 50 mL of water were added instead) and the reaction e was extracted with ethyl acetate (3x20 mL). United ethyl acetate fractions were washed with water (1x10 mL) dried over MgSO4 and evaporated to give crude product.
Products were purified by flash chromatography (gradient of 20 minutes from 100% of cyclohexane to 100% of ethyl acetate).
B. To a degased solution of the proper aryl halide (1 eq., 1 mmol) in THF (5 mL) and TEA (5 mL), premixed PPh3)2 (0.03 eq., 30 umol) and Cu] (0.06 eq., 60 umol) were added, followed by the addition of propargyl alcohol (2 eq., 1.2 mmol). The reaction mass was stirred for 1-24 hours (monitored by TLC). THF and TEA were evaporated and the crude product was purified by flash chromatography (gradient of 20 minutes from 100% of cyclohexane to 100% of ethyl acetate).
C. To a degased solution of the proper aryl halide (1 eq., 1 mmol) in propylamine or pyrrolidine (3 mL), Pd(PPh3)4 (0.05 eq., 50 umol) was added. The reaction mass was heated ght (30-50 °C), evaporated and the crude product was purified by flash tography (gradient of 20 minutes from 100% of cyclohexane to 100% of ethyl acetate).
WO 20151001117 31 Preparation of highly active MnOz A solution of MnC12'4H20 (1 eq., 1 mole, 200 g) in water (2 L) at 70 0C was gradually added during 10 minutes, with stirring, to a solution of KMnO4 (1 eq., 1 mole, 160 g) in water (2 L) at 60 °C in a hood. A vigorous reaction ensued with ion of chlorine; the sion was stirred for 2 hours and kept overnight at room temperature. The precipitate was filtered off, washed thoroughly with water (4 L) until pH 6.5-7 and the washing gave a negligible chloride test. The filter cake was then dried at 120—130 °C for 18 h; this gave a chocolate-brown, highly disperse amorphous powder.
MnOz oxidation OH R1 / R2 // Slightly modified procedure described by McAllister 824 To et al. a solution of the proper propargylic alcohol (1 eq., 1 mmol) in THF (4.5 mL), MgSO4 (15 eq., 15 mmol), highly active Mn02 (25 eq., 25 mmol) and 2M NHs on in IPA (4 eq., 4 mmol, 2 mL) were added. Obtained reaction mass was usly stirred at room temperature for 05-12 hours (monitored by TLC). DCM (20 mL) were added and the obtained on mass was filtered through Celite, evaporated to give crude product and purified by flash chromatography if required.
WO 2015001117 32 2014/064387 Substituted 3-(aryl)propynols (la-12a): R3 R4 R3 R4 1a-12a R1 R2 R3 R4 X Protocol 1a OMe H H H I B 2a H OMe H H Br C 3a H H OMe H I B 4a OMe H H OMe I C 5a NH2 H H H I B 6a H NH2 H H I A 7a H H NH2 H I A 8a Me H H H I B 9a Me H H Me I A 10a N02 H H H Br B 11a H H NHAc H I A 12a H H CONHMe H I A 3-(2-Methoxyphenyl)propyn-l-ol (la).
Reaction time: 18 hours; yield: 72%. 1H NMR (400 MHz, METHANOL-d4) 8 7.36 (dd, J = 1.5, 7.5 Hz, 1H), 7.26-7.33 (m, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.86-6.93 (m, 1H), 4.43 (s, 2H), 3.84 (s, 3H); 13C NMR (101 MHz, METHANOL-d4) 5161.6, 134.5, 131.0, 121.5, 113.4, 112.0, 92.7, 82.0, 56.2, 51.5. 3-(3-Methoxyphenyl)propynol (23).
Reaction time: 16 hours; yield: 87%. 1H NMR (400 MHz, METHANOL-d4) 5 7.15-7.24 o-t, J = 7.5 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.93-6.98 (m, 1H), 6.87 (dd, J: 2.13, 7.5 Hz, 1H), 4.41 (s, 2H), 3.73 (s, 3H); 13C NMR (101 MHz, METHANOL-d4) 5 160.9, 130.6, 125.4, 125.1, 117.8, 115.7, 88.8, 85.6, 55.9, 51.4. 3-(4-Methoxyphenyl)propynol (33).
Reaction time: 16 hours; yield: 92%.
WO 2015001117 33 1H NMR (400 MHz, CHLOROFORM-d) 8 7.40 (d, J = 8.78 Hz, 2H), 6.86 (d, J = 8.78 Hz, 2H), 4.51 (d, J: 4.9 Hz, 2H), 3.83 (s, 3H), 1.78 (t, J: 4.9 Hz, 1H); 13C NMR (101 MHz,CHLOROFORM-d)8159.8, 133.2, 114.6, 114.0, 85.9, 85.7, 55.3, 51.7. 3-(2-Aminophenyl)propynol (43). on time: 24 hours; yield: 62%. 1H NMR (400 MHz, METHANOL-d4) 8 7.19 (dd, J = 1.25, 7.9 Hz, 1H), 7.03-7.12 (m, 1H), 6.75 (d, J = 7.9 Hz, 1H), 6.56-6.65 (m, 1H), 4.47 (s, 2H), 4.26 (s, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 150.3, 133.0, 130.6, 118.2, 115.6, 93.8, 78.9, 69.5, 51.0. 3-(3-Aminophenyl)propyn-l-ol (53).
Reaction time: 18 hours; yield: 77%. 1H NMR (400 MHz, FORM-d) 8 7.08 (t, J= 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.64 (dd, J = 1.5, 7.8 Hz, 1H), 4.46 (s, 2H), 2.17 (s, 1H); 13C NMR (101 MHz, CHLOROFORM-d) 8 146.3, 129.2, 123.4, 122.0, 118.0, 115.5, 87.0, 85.6, 51.4. 3-(4-Aminophenyl)propyn01 (63).
Reaction time: 18 hours; yield: 42%. 1H NMR (400 MHz, METHANOL-d4) 8 7.11-7.21 (d, J: 8.5 Hz, 2H), 6.53-6.68 (d, J: 8.5 Hz, 2H), 4.37 (s, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 149.7, 133.8, 115.7, 112.5, 86.7, 85.9, 51.4; 3-(2-Nitrophenyl)propynol (73).
Reaction time: 15 hours; yield: 35%. 1H NMR (400 MHz, CHLOROFORM-d) 8 7.97 (d, J = 8.0 Hz, 1H), 7.54—7.60 (d, J = 8.0 Hz, 1H), 7.46-7.54 (t, J = 8.0 Hz, 1H), 7.36-7.44 (t, J = 8.0 Hz, 1H), 4.49 (s, 2H), 1.68 (br. s., 1H); 13C NMR (101 MHz, CHLOROFORM-d) 8 149.9, 134.8, 132.8, 128.9, 124.6, 118.0, 95.2, 80.9, 51.7. 4-(3-Hydroxypropynyl)-N-methylbenzamide (83). on time: 12 hours; yield: 91%. 1H NMR (400 MHz, METHANOL-d4) 8 7.72-7.82 (m, J = 8.28 Hz, 2H), 7.41-7.53 (m, J = 8.28 Hz, 2H), 4.43 (s, 2H), 2.92 (s, 3H); 13C NMR (101 MHz, METHANOL—d4) 8 169.9,135.2,132.7, 128.3, 127.6, 91.5, 84.7, 51.3, 27.1.
N-(4-(3-Hydroxypropynyl)phenyl)acetamide (93).
Reaction time: 18 hours; yield: 85%.
WO 2015001117 34 1H NMR (400 MHz, METHANOL-d4) 8 7.56-7.64 (d, J = 8.8 Hz, 2H), 7.47-7.56 (d, J = 8.8 Hz, 2H), 4.74 (s, 2H), 2.04 (s, 3H); 13C NMR (101 MHz, METHANOL-d4) 8 171.9, 143.8, 135.7, 120.7, 112.8, 106.2, 84.5, 62.7, 24.1. 3-(0-Tolyl)propynol (1021).
Reaction time: 5 hours; yield: 70%. 1H NMR (400 MHz, CHLOROFORM-d) 6 7.40 (d, J = 7.5 Hz, 1H), .24 (m, 3H), 4.54 (s, 2H), 2.43 (s., 3H); 13C NMR (101 MHz, CHLOROFORM-d) 8 138.2, 131.2, 128.4, 128.0, 119.3, 115.3, 86.5, 85.2, 51.2, 21.2. 3-(2,6-Dimethylphenyl)propynol (lla).
Reaction time: 24 hours; yield: 25%. 1H NMR (400 MHz, CHLOROFORM-d) 8 7.03 (t, J= 7.5 Hz, 1H), 6.95 (d, J = 7.5 Hz, 2H), 4.50 (s, 2H), 2.34 (s, 7H); 13C NMR (101 MHz, CHLOROFORM—d) 8 140.5, 127.9, 126.7, 122.3, 95.6, 83.3, 51.9, 21.1.
-Dimethoxyphenyl)propynol (12a).
Reaction conditions: 30 °C, propylanline, 16 hours; yield: 38%. 1H NMR (400 MHz, METHANOL-d4) 8 7.25 (t, J = 8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 4.46 (s, 2H), 3.84 (s, 6H); 13C NMR (101 MHz, METHANOL—d4) 8 163.0, 131.0, 104.7, 102.5, 97.0, 78.1, 56.4, 51.7.
WO 01117 35 Substituted 3-aryl-pr0piolonitriles (1-12): R, R1 / R2 // MnOZY Mgso4 R2 // NH3 (IPA), THF R3 R4 R3 R4 R1 R2 R3 R4 Time, h Yield, 0/0 1 OMe H H 3 45 2 H OMe H 2 85 3 H OMe H 3 95 4 OMe H H OMe 4 60 NH2 H H 1 47 6 H NH2 H 2 71 7 H H NH2 9 94 8 Me H H 1.5 70 9 Me H H Me 2 55 N02 H H H 4 21 11 H H NHAC H 2 92 12 H H CONHMe H 2 61 3-(2-Methoxyphenyl)pr0piolonitrile (1, APN-o-OMe). 1H NMR (400 MHz, METHANOL-d4) 8 7.51-7.65 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 7.01 (t, J = 7.7 Hz, 1H), 3.94 (s, 4H); 13C NMR (101 MHz, METHANOL—d4) 8 164.7, 136.4, 135.3, 122.0, 112.6, 107.7, 106.4, 81.8, 66.7, 56.7; IR (neat film, cm'l): 2946, 2264, 2142 1596, 1490, 1245, 1164, 1122, 1047, 1021, 752, 498; GC-ESI-HRMS: 157.05276; found 157.05044. 3-(3-Methoxyphenyl)pr0piolonitrile (2, APN-m-OMe). 1H NMR (400 MHz, METHANOL-d4) 8 7.38 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.20-7.24 (m, 1H), 7.12-7.20 (m, 1H), 3.83 (s, 3H); 13C NMR (101 MHz, METHANOL—d4) 8 161.2, 131.4, 127.1, 120.0, 119.4, 119.2, 106.0, 84.1, 62.7, 56.1; IR (neat film, cm‘l): 2491, 2264, 2144, 1595, 1573, 1488, 1464, 1420, 1324, 1294, 1207, 1178, 1045, 783, 681, 494; GC-ESI-HRMS: 276; found 157.05298. 3-(4-Methoxyphenyl)pr0piolonitrile (3, APN-p-OMe). wo 2015;001117 36 1H NMR (400 MHz, CHLOROFORM-d) 8 0 (m, J = 8. 8 Hz, 2H), 6.86-6.96 (m, J = 8.8 Hz, 2H), 3.86 (s, 3H); 13C NMR (101 MHz, CHLOROFORM-d) 8 161.4, 134.4, 113.7, 108.2, 104.8, 82.7, 61.5, 54.5; IR (neat film, cm'l): 2985, 2358, 2342, 2263, 2178, 2149, 1603, 1514, 1307, 1270, 1180, 1028, 835, 808, 669, 424; GC-ESI- HRMS: 157.05276; found 157.05337. 3-(2,6-Dimethoxyphenyl)propiolonitrile (4, AFN-0,0 ’-diOMe): 1H NMR (400 MHz, CHLOROFORM—d) 8 7.38 (t, J: 8.5 Hz, 1H), 6.53 (d, J = 8.5 Hz, 2H), 3.88 (s, 6H); 13C NMR (101 MHz, CHLOROFORM-d) 8 164.4, 133.8, 106.2, 103.4, 96.5, 77.7, 70.5, 562; IR (neat film, cm'l): 2847, 2359, 2259, 2201, 2139, 1926, 1586, 1574, 1478, 1432, 1302, 1255, 1188, 1109, 1025, 778, 727, 648, 632, 545, 506, 488, 420; GC-ESI-HRMS: 187.06333; found 184.06465. 3-(2-Aminophenyl)propiolonitrile (5, APN-o-NHz). 1H NMR (500 MHz, METHANOL-d4) 8 6.81 (d, J = 7.88 Hz, 1H), 6.65-6.76 (m, 1H), 6.08-6.19 (m, 2H), 3.85 (br. s., 1H); 13C NMR (126 MHz, CHLOROFORM-d) 8 151.4, 134.0, 133.4, 118.2, 115.0, 105.8, 101.0, 81.6, 68.5; IR (neat film, cm'l): 3413, 3332, 3211,2925, 2853,2250, 2136, 1632, 1600, 1563, 1486, 1452, 1312, 1273, 1252, 1161, 740, 673, 493; GC-ESI-HRMS: 310; found 142.05458. 3-(3-Aminophenyl)propiolonitrile (6, APN-m-NHz). 1H NMR (400 MHz, CHLOROFORM-d) 8 7.17 (t, J: 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.74-6.89 (m, 2H), 3.85 (br. s., 2H); 13C NMR (101 MHz, CHLOROFORM-d) 5 146.8, 129.8, 123.6, 118.7, 118.7, 118.0, 105.7, 83.7, 623; IR (neat film, cm'l): 3426, 3340, 2923, 2852, 2265, 2142, 1630, 1594, 1579, 1513, 1448, 1326, 1313, 1300, 1220, 1164, 993, 882, 862, 784, 680, 534, 456; GC—ESI-HRMS: 142.05310; found 142.05197. 3-(4-Aminophenyl)propiolonitrile (7, APN-p-NHz). 1H NMR (400 MHz, METHANOL-d4) 5 7.26 (d, J = 8.6 Hz, 2H), 6.51 (d, J = 8.6 Hz, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 152.5, 135.1, 113.6, 105.6, 102.3, 86.3, 60.2; IR (neat film, cm‘l): 3431, 3333, 3211,2250, 2132, 1632, 1599, 1513, 1438, 1303, 1178, 1043, 949, 826, 814, 526, 495, 452; -HRMS: 142.05310; found 142.05464. 3-(0-Tolyl)propiolonitrile (8, APN-o-Me). 1H NMR (400 MHz, CHLOROFORM-d) 8 7.47 (d, J = 7.78 Hz, 1H), 7.28-7.36 (m, 1H), 7.18 (d, J = 8.03 Hz, 1H), 7.08-7.16 (m, 1H), 2.39 (s, 3H); 13C NMR (101 MHz, W0 20151001117 37 CHLOROFORM-d) 8 143.4, 134.1, 131.8, 130.1, 126.1, 117.4, 105.6, 82.4, 66.4, 20.5; IR (neat film, cm'l): 2295, 2257, 2141, 1599, 1484, 1456, 1383, 1291, 1199, 1162, 1116, 1039, 757, 711, 672, 548, 490, 452; GC-ESI-HRMS: 141.05785; found 141.05926. 3-(2,6-Dimethylphenyl)propiolonitrile (9, APN-o,0’-diMe). 1H NMR (400 MHz, CHLOROFORM-d) 8 7.12-7.27 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 7.5 Hz, 2H), 2.38 (s, 6H); 13C NMR (101 MHz, CHLOROFORM-d) 8 143.8, 131.2, 127.3, 117.6, 105.6, 81.5, 70.2, 20.8; IR (neat film, cm'l): 2923, 2856, 2261, 2138, 1732, 1595, 1468, 1381, 1265, 1168, 1033, 774, 728, 490; GC-ESI-HRMS: 15507350; found 155.07507. 3-(2-Nitrophenyl)propiolonitrile (10, APN-o-NOz). 1H NMR (400 MHz, METHANOL-d4) 8 8.28 — 8.35 (m, 1H), 7.96 — 8.06 (m, 1H), 7.81 — 7.90 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 151.9, 138.3, 135.2, 134.1, 126.6, 114.2, 105.7, 79.0, 68.6; IR (neat film, cm‘l): 2268, 1604, 1567, 1528, 1502, 1480, 1345, 851, 787, 744, 709, 687, 537, 491; GC-ESI-HRMS: 172.02728; found 172.02869.
N-(4-(Cyanoethynyl)phenyl)acetamide (11, APN-p-NHAc). 1H NMR (400 MHz, METHANOL—d4) 5 .63 (m, J = 8.8 Hz, 2H), 7.49-7.56 (m, J = 8.8 Hz, 2H), 2.04 (s, 3H); 13C NMR (101 MHz, METHANOL—d4) 8 171.9, 143.8, 135.7, 120.7, 112.8, 106.2, 84.5, 62.7, 24.1; IR (neat film, cm‘l): 3303, 3174, 3098, 2278, 2262, 2139, 1670, 1594, 1535, 1407, 1364, 1321, 1263, 1177, 834, 534; GC-ESI- HRMS: 184.06366; found 184.06212. 4-(Cyanoethynyl)-N-methylbenzamide (12, CONHMe): 1H NMR (400 MHz, METHANOL-d4) 8 7.96-8.05 (m, J = 7.78 Hz, 2H), 7.85-7.93 (m, J = 7.78 Hz, 2H), 3.03 (s, 3H); 13C NMR (101 MHz, OL-d4) 8 169.1, 138.3, 134.9, 129.0, 121.6, 105.9, 83.0, 64.6, 28.8; IR (neat film, cm'l): 3348, 2270, 1641, 1549, 1502, 1408, 1392, 1327, 1303, 1283, 1162, 854, 760, 617, 488; GC-ESI-HRMS: 184.06366; found 184.06465.
WO 20151001117 38 3-(4-10d0phenyl)propiolonitrile (13): é OH / MnOz, Mgso4 é NH3/IPA/THF | r.t., 2h I Product was synthesized according to general procedure of MnOz-oxidation. Reaction time: 30 minutes; yield: 61%. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.78 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 3H); 13C NMR (101 MHz, chloroform-d) 8 138.1, 134.4, 116.8, 105.2, 99.2, 81.9, 64.2.
Compound 13 can be used for the ng method according to the invention (with radioisotope 1251). 3-(4-(Triflu0r0methyl)phenyl)pr0piolonitrile (14): é OH / Mn02,MgSO4 é NH3/IPNTHF F3C r.t.,2h F3C Product was synthesized ing to general procedure of MnOz-oxidation. Reaction time: 1 hour; yield: 45%. 1H NMR (400MHz, CHLOROFORM-d) 5 = 7.76 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H).
Compound 14 can be used for the labeling method according to the invention (with radioisotope 18F). tert-Butyl 4-(3-hydr0xypr0pynyl)benz0ate (15a): Refri— XorC/OH t was synthesized according to l procedure B for coupling. Yield : 98%.
WO 2015001117 39 1H NMR (400MHz, CHLOROFORM-d) 5 = 7.93 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 4.53 (s, 2H), 1.60 (s, 9H); 13C NMR (101MHz, CHLOROFORM-d) 5 = 165.1, 131.7, 131.4, 129.3, 126.6, 89.8, 85.1, 81.4, 51.6, 28.1. tert-Butyl 4-(cyanoethynyl)benz0ate (15): / // / Mno2 Mgso4 ¢ NH3/IPtA/THF X0 Product was synthesized according to general procedure of MnOz-oxidation. Reaction time: 15 minutes; yield: 48%. 1H NMR (400MHz, DMSO-ds) 5 = 8.00 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 1.56 (s, 9H); 13C NMR (101MHz, FORM-d) 5 = 164.3, 134.8, 133.3, 129.7, 121.3, 105.2, 82.2, 81.9, 64.8, 28.1. noethynyl)benz0ic acid (16): //N //N ¢ ¢ X0 ’ o 0 To the solution of tert-butyl 4-(2-cyanoeth—I-yn-I—yl)benz0ate (1 eq., 350 mg, 1.54 mmol) in MeCN (14 mL) was added TFA (30.6 eq., 5.372 g, 3.5 mL, 47.1 mmol) . The mixture was stirred for 36h at r.t. and then filtered and washed with 3X2 mL of EtzO.
The precipitate consisted of pure 4-(2-cyanoeth-I-yn-I-yl)benz0ic acid (140 mg, 0.823 mmol, 53% . 1H NMR (400MHz, METHANOL-d4) 8 = 8.12 (d, J = 8.3 Hz, 3H), 7.83 (d, J = 8.3 Hz, 2H).
Perfluorophenyl 4-(cyan0ethynyl)benz0ate (1 7): WO 01117 40 16 17 The solution of pentafluorophenol (1 eq., 89.2 mg, 0.484 mmol) and 4-(2—cyanoeth yny1)benzoic acid (1 eq., 82.9 mg, 0.484 mmol) in THF (4.84 mL) was cooled to 0°C and DCC (1 eq., 99.9 mg, 0.484 mmol) was added to the mixture. The ing solution was d at r.t. for 14h, then filtered and washed with EtzO. The filtrate was evaporated to give pentafluorophenyl 4—(2-cyanoethyny1)benzoate (120 mg, 0.358 mmol, 74% yield) as a white solid. 1H NMR z, DMSO-d6) 5 = 8.29 (d, J = 8.3 Hz, 2H), 8.08 (d, J = 8.3 Hz, 2H).
Compound 17 can be used for a bio-conjugation method according to the invention.
Sodium 4-((4-(cyanoethynyl)benz0yl)0xy)-2,3,5,6-tetraflu0r0benzenesulfonate (18): 4” ¢ ¢ F NaO3S F 16 18 To the solution of 4-(2—cyanoethyn—1-yl)benzoic acid (1 eq., 54.2 mg, 0.317 mmol) and sodium 2,3,5,6-tetrafluorohydroxybenzenesulfonate (1 eq., 84.9 mg, 0.317 mmol) in dry DMF (0.792 mL) was added DCC (1 eq., 65.3 mg, 0.317 mmol) . The resulting mixture was stirred at r.t. for 36h, then cooled to 0°C, stirred for 1h, filtered and washed with 0.8 mL of dry DMF. The filtrate was diluted with 16 mL of EtzO, stirred for 15 min for complete crystallization and the precipitate was filtered to give sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3 ,5 ,6-tetrafluorobenzenesulfonate (72 .5 mg, 0.172 mmol, 54% yield) as a white solid. 1H NMR (400MHz, DMSO-ds) 8 = 8.31 (d, J = 6.3 Hz, 2H), 8.09 (d, J = 6.3 Hz, 2H).
Compound 18 can be used for a bio—conjugation method according to the invention.
WO 2015001117 41 N-((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triaz0lyl)methyl) (dimethylamin0)naphthalenesulf0namide (20): N” o o f: N.]/\Hw o Og-NH 0 II —> + N \ o / || N // 19 N 20 ethylamino)-N-(propynyl)naphthalenesulfonamide (1 eq., 395 mg, 1.37 mmol) and 3—(4-azidophenyl)prop-2—ynenitrile (1 eq., 230 mg, 1.37 mmol) were solubilized in tBuOH (6.91 mL). To this mixture was added a solution of copper sulfate pentahydrate (10 %, 34.2 mg, 0.137 mmol) in 0.5 mL of water followed by the solution of sodium ascorbate (0.5 eq., 135 mg, 0.685 mmol) in 0.5 mL of water. The resulting solution was stirred for 2h and then concentrated on rotary evaporator. The residue was extracted with DCM. The organic layer was washed with saturated s solution of NH4C1 and with water, dried over MgSO4 and evaporated to give 20 (544 mg, 1.19 mrnol, 87% yield) as a green solid. 1H NMR (400MHz, DMSO-ds) 5 = 8.59 (br. s., 1H), 8.34 (d, J = 7.3 Hz, 1H), 8.33 (s, 1H) 8.26 (d, J: 8.8 Hz, 1H), 8.13 (d, J= 7.3 Hz, 1H), 8.00 (d, J: 8.8 Hz, 2H), 7.83 (d, J: 8.8 Hz, 2H), 7.61 — 7.51 (m, 2H), 7.18 (d, J: 7.5 Hz, 1H), 4.21 (s, 2H), 2.71 (s, 6H). 13C NMR z, DMSO-ds) 8 = 151.2, 144.8, 138.4, 135.7, 135.6, 129.4, 128.9, 128.8, 128.6, 127.8, 123.4, 121.3, 119.9, 119.0, 116.0, 114.9, 105.3, 82.5, 63.1, 44.9, 37.6.
Compound 20 can be used for a detection method (with dyes, for instance) according to the invention. 3-(4-is0thiocyanat0phenyl)propiolonitrile (21): WO 20151001117 42 //N //N é i Cl C] é NaHCO3, DCM/H20 Silt H2” N 0°C, 10 min In a 50 mL RB flask, a solution of sodium hydrogen carbonate (886 mg, 10.55 mmol) in mL water was stirred for 10 min and to it dichloromethane (10 mL) was added followed by 3-(4-amin0phenyl)pr0pynenitrile (500 mg, 3.52 mmol). The reaction mixture was cooled to 0°C, thiophosgene (402 uL, 5.28 mmol) was introduced dropwise over a period of 30 min and continuously d at room temperature for 1 h.
The organic phase was ted and dried over ous MgSO4. Concentration of the solution afforded pure 21 (609 mg, 3.31 mmol, 94% yield) in form of yellow solid. 1H NMR (400MHz, ACETONITRILE-ds) 8 = 7.71 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H).
Compound 21 can be used for a bio-conjugation method according to the invention. tert-butyl (1-((4-(cyan0ethynyl)phenyl)amin0)thi0x0-6,9,12-tri0xa azapentadecanyl)carbamate (22): S o & NJLNMO/Vowo/WNAOk // H H H 21 22 tert-Butyl (3-(2-(2-(3—amin0pr0p0xy)ethoxy)eth0xy)pr0pyl)carbamate (1 eq., 91.5 mg, 0.271 mmol) was dissolved in of DCM (2 mL) and cooled to 0°C. To this solution 3-(4- ocyanatophenyl)pr0pynenitrile (1 eq., 50 mg, 0.271 mmol) in 1 mL of DCM was slowly added and the mixture was stirred for 30 min. The reaction mix was concentrated to 1 mL and the residue was purified by flash chromatography (DCM/MeOH nt, 100/0 to 90/10) to give tert-butyl (1-((4- (cyanoethynyl)phenyl)amin0)—I -thi0x0-6, 9, 12—tri0xa—2-azapentadecan-I5-yl)carbamate (126 mg, 0.25 mmol, 92% yield) as a yellow oil.
WO 20151001117 43 1H NMR (400MHz, OL-d4) 5 = 7.66 (s, 4H), 3.65 — 3.54 (m, 12H), 3.50 (t, J = 6.1 Hz, 2H), 3.12 (t, J = 6.8 Hz, 2H), 1.91 (quin, J = 6.1 Hz, 2H), 1.72 (quin, J = 6.4 Hz, 2H), 1.45 (s, 9H). 13c NMR (101MHz, METHANOL—d4) 5 = 182.0, 158.5, 144.6, 135.6, 106.4, 84.8, 80.0, 71.6, 71.5, 71.3, 71.3, 70.0, 68.2, 63.0, 38.8, 31.0, 29.8, 29.0 4-(cyan0ethynyl)benz0yl chloride (23): N\\ N\\ % % OH CI 16 23 yanoeth-I-yn-I—yl)benzoic acid (1 eq., 30 mg, 0.175 mmol) was dissolved in DCM (2 mL) and SOC12 (31.5 eq., 400 uL, 5.51 mmol) was added. The mixture was stirred at reflux until the solid completely dissolved and then evaporated to give pure 23 (29.6 mg, 0.156 mmol, 89% yield) as a white solid. 1H NMR (400MHz, CHLOROFORM-d) 8 = 8.08 (d, J = 8.3 Hz, 2H), 7.67 (d, J: 8.3 Hz, 2H). nd 23 can be used for a bio-conjugation method according to the invention. tert-butyl (1-(4-(cyanoethynyl)phenyl)0x0-6,9,12-tri0xaazapentadecan yl)carbamate (24): N \ \ \ Q —> N\/\/ \/\O/\/ \/\/O O H 0 CI \n/ \'< O O 23 24 tert-Bulyl (3 —(2-(2-(3-amin0pr0p0xy)ethoxy)ethoxy)propyl)carbamate (1 eq., 50 mg, 0.156 mmol) and NEt3 (5 eq., 78.9 mg, 0.108 mL, 0.78 mmol) were dissolved in 1 mL of DCM and cooled to -78°C. To this solution was slowly added 23 (1 eq., 29.6 mg, 0.156 mmol) in 1 mL of DCM. The mixture was gradualy warmed to r.t. and stirred for WO 20151001117 44 2h. The reaction mix was then injected into flash chromatography column and eluted with DCM/MeOH (gradient 100/0 to 90/10) to give pure 24 (40.6 mg, 0.086 mmol, 55 %) as a yellow oil. 1H NMR (400MHz, METHANOL-d4) 8 = 7.92 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 3.72 - 3.45 (m, 14H), 3.12 (t, J = 6.8 Hz, 2H), 1.90 (quin, J = 6.3 Hz, 2H), 1.72 (quin, J = 6.4 Hz, 2H), 1.44 (s, 9H). 4-(cyan0ethynyl)-N-(15-0x0-4, 7,10-tri0xaazan0natriaconta-24,26-diyn yl)benzamide (25): 16 ~\\\ 0 ‘\—0 NH 4-(2-cyanoethynyl)benzoic acid (1 eq., 29.7 mg, 0.173 mmol) was suspended in DCM and SOClz (39.8 eq., 820 mg, 0.5 mL, 6.89 mmol) was added. The mixture was d at reflux for 1.5h, evaporated, dissolved in DCM, and added to the solution of N- (3 - {2-[2-(3 propoxy)ethoxy]ethoxy}propyl)pentacosa-10, 12-diynamide (1 eq. , 100 mg, 0.173 mmol) and TEA (4 eq., 70.2 mg, 0.0964 mL, 0.693 mmol) in DCM at -78°C. The resulting mixture was stirred at r.t. for 1h and evaporated. The residue was purified by flash chromatography (DCM/MeOH : 10/0 to 9/ 1) to give the desired product (35.4 mg, 0.0485 mmol, 28% yield) as a yellow 011. 1H NMR z, CHLOROFORM—d) 8 = 7.90 (d, J = 8.3 Hz, 2H), 7.67 (d, J: 8.3 Hz, 2H), 7.48 (br. s, 1H), 6.18 (br. s, 1H), 3.71 - 3.45 (m, 14H), 3.32 (t, J: 6.0 Hz, 2H), 2.23 (t, J: 6.8 Hz, 4H), 2.15 (t, J = 7.5 Hz, 2H), 1.90 (td, J: 6.0, 11.7 Hz, 2H), 1.73 (quin, J: 6.1 Hz, 2H), 1.65 - 1.42 (m, 6H), 1.41 - 1.32 (m, 4H), 1.32 - 1.19 (m, 22H), 0.88 (t, J= 6.8 Hz, 3H). nd 25 can be used for the labeling (such as photolabeling) method according to the invention or for binding and/or immobilizing compounds.
WO 20151001117 45 Ethyl (4-(cyan0ethynyl)phenyl)carbamate (26): 16 26 To a solution of triphosgene (1 eq., 49.5 mg, 27.8 uL, 0.167 mmol) in DCM (4 mL) was added a solution of 3-(4-aminophenyl)propynenitrile (3 eq., 71.1 mg, 0.5 mmol) in DCM (1 mL). Then triethylamine (6 eq., 101 mg, 138 uL, 1 mmol) in 1 mL of DCM was added dropwise. The mixture was stirred for 15 min allowing the formation of isocyanate intermediate and then ethanol (0.1 mL) was added dropwise. The reaction mixture was stirred for 1h then washed with 2x5mL of water and evaporated. The residue was d by flash chromatography to give ethyl (4- ethynyl)phenyl)carbamate (102 mg, 0.48 mmol, 96% yield) as a white solid. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.56 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 6.79 (br. s., 1H), 4.26 (q, J= 7.0 Hz, 2H), 1.33 (t, J= 7.0 Hz, 3H). 1-(4-(cyanoethynyl)phenyl)(propynyl)urea (27): N //N // //N é é é —’ —’ o\ i \ N N H2N C“N /H H 7 27 To a solution of triphosgene (1 eq., 49.5 mg, 27.8 uL, 0.167 mmol) in DCM (2 mL) was added a solution of 3—(4-aminophenyl)propynenitrile (3 eq., 71.1 mg, 0.5 mmol) in DCM (3 mL). Then triethylamine (6 eq., 101 mg, 138 uL, 1 mmol) was added, the mixture was d for 5 min and then were added propargylamine (4.69 eq., 43 mg, 50.1 uL, 0.782 mmol) and triethylamine (2 eq., 33.7 mg, 46.3 uL, 0.333 mmol) in 1 mL of DCM. The reaction mixture was stirred for 1h then washed with 5mL of water, dried over MgSO4 and concentrated. The residue was purified by flash chromatography eOH gradient) to give l-(4-(cyanoethynyl)phenyl)-3—(prop—2-yn-l—yl)urea (94.9 mg, 0.425 mmol, 85% yield) as a white solid.
WO 20151001117 46 1H NMR (400MHz, METHANOL-d4) 5 = 7.60 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 4.00 (d, J = 2.4 Hz, 2H), 2.61 (t, J = 2.4 Hz, 1H).
Compound 27 can be used for click chemistry according to the invention. prop-Z-yn-I-yl (4-(cyan0ethynyl)phenyl)carbamate (28): To a solution of triphosgene (1 eq., 49.5 mg, 27.8 uL, 0.167 mmol) in DCM (2 mL) was added a solution of 3-(4-aminophenyl)prop—2-ynenitrile (3 eq., 71.1 mg, 0.5 mmol) in DCM (3 mL). Then triethylamine (6 eq., 101 mg, 138 uL, 1 mmol) was added. The mixture was stirred for 5 min and then were added ynol (6 eq., 56.1 mg, 59.1 11L, 1 mmol) and triethylamine (2 eq., 33.7 mg, 46.3 uL, 0.333 mmol) in 1 mL ofDCM.
The reaction mixture was stirred for 1h then washed with 5mL of water, dried over MgSO4 and concentrated. The e was purified by flash chromatography (Cyclohexane/EtOAc gradient) to give prop—2-yn-I —yl (4- (cyanoethynyl)phenyl)carbamate (104 mg, 0.465 mmol, 93% yield) as a white solid. 1H NMR (400MHz, CHLOROFORM—d) 8 = 7.57 (d, J = 8.6 Hz, 2H), 7.48 (d, J: 8.6 Hz, 2H), 4.80 (d, J: 2.3 Hz, 2H), 2.54 (t, J: 2.3 Hz, 1H).
Compound 28 can be used for click chemistry according to the invention. bicycl0[6.1. 4-ynylmethyl (4-(cyan0ethynyl)phenyl)carbamate (29): _, OJL” 7 29 To a solution of triphosgene (1 eq., 34.8 mg, 19.5 uL, 0.117 mmol) in DCM (4 mL) was added a solution of 3-(4-aminophenyl)propynenitrile (3 eq., 50 mg, 0.352 mmol) in DCM (1 mL). Then triethylamine (6 eq., 71.2 mg, 97.8 uL, 0.703 mmol) was added W0 20151001117 47 dropwise. The mixture was stirred for 5 min at r.t. and then bicyclo[6.l.0]nonyn ylmethanol (3 eq., 52.8 mg, 0.352 mmol) and triethylamine (2 eq., 23.7 mg, 32.6 uL, 0.234 mmol) were added in 1 mL of DCM. The reaction mixture was stirred at r.t. for 2 hours. After full conversion was confirmed by HPLC the mixture was concentrated to 1 mL volume and purified by flash chromatography (cyclohexane/EtOAc gradient) to give bicyclo[6.l.0]nonyn—9-ylmethyl (4-(cyanoethynyl)phenyl)carbamate (68.3 mg, 0.215 mmol, 183 %) as a white solid. 1H NMR z, CHLOROFORM-d) 8 = 7.55 (d, J = 8.8 Hz, 2H), 7.47 (d, J: 8.8 Hz, 2H), 7.09 (br. s, 1H), 2.38 - 2.14 (m, 6H), 1.67 - 1.51 (m, 2H), 1.42 (quin, J: 8.7 Hz, 1H), 1.04 - 0.91 (m, 2H) nd 29 can be used for click chemistry (such as strain-promoted click) according to the invention. (trimethylsilyl)ethynyl)phenyl)pr0pyn0l (30a): /©/| / >Si \ /<j/\QHé I /S||_ 303 Product was synthesized according to general procedure B for coupling. Yield : 99%. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.41 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 4.50 (d, J= 5.5 Hz, 2H), 1.89 (t, J= 5.5 Hz, 1H), 0.25 (s, 9H). 3-(4-(trimethylszlyl)ethynyl)phenyl)pr0p10l0nltrlle (30): 30a 30 Product was sized according to general procedure of MnOz-oxidation. Reaction time: 3 hours; yield: 29%. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.55 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 0.27 (s, 9H).
WO 2015001117 48 13C NMR (101MHz, FORM-d) 8 = 133.2, 132.2, 126.9, 117.1, 105.3, 103.4, 99.4, 82.3, 64.5, -0.3. 3,3 '-(5-amin0-I,3-phenylene)dipr0piolonitrile (31): Product was synthesized according to general procedure of MnOz-oxidation. Reaction time: 3 hours; yield: 11%. 1H NMR (400MHz, CHLOROFORM-d) 5 = 7.20 (t, J = 1.3 Hz, 1H), 6.98 (d, J = 1.3 Hz, 2H), 4.02 (br. s., 2H). 13C NMR (101MHz, CHLOROFORM-d) 8 = 147.1, 127.5, 121.6, 119.5, 105.0, 81.0, 63.6. nd 31 can be used for rebridging (diAPN) according to the invention. 3-(4-(4, 4,5,5-tetramethyl-1,3,2-di0xab0rolanyl)phenyl)propiolonitrile (32): D// OH O\ —> '13 /[:/ O 0‘s Product was synthesized according to general procedure of MnOz—oxidation. on time: 4 hours; yield: 63%. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.84 (d, J = 8.2 Hz, 2H), 7.60 (d, J: 8.2 Hz, 2H), 1.36 (s, 12H). 3,3'-(1,2-Phenylene)dipropiolonitrile (33): propargylic / I / alcoho|,Cu|, // PdCI2(PPh3)2I OH Mn02, NHs I —> — —> DIPEA, DMF M9804, IPA, THF \ 33a 33 333: 3,3'-(1,2-Phenylene)bis(propyn-l-ol). wo 20157001117 49 To the degased solution of 1,2-diiodobenzene (1 eq., 661 mg, 0.262 mL, 2 mmol) and propargylic alcohol (2.3 eq., 272 uL, 4.61 mmol) in butyl amine (15.8 mL), Pd(PPh3)4 (4%, 92.6 mg, 0.0801 mmol) was added and the obtained reaction mass was refluxed ght. ts were ated and the obtained crude product was purified by flash chromatography (20 minutes gradient EtOAc/Cyclohexane) to yield 333 (150 mg, 0.8 mmol, 40 %) as a brownish solid. 1H NMR (400 MHz, METHANOL-d4) 8 7.38 - 7.53 (m, 2H), 7.25 — 7.38 (m, 2H), 4.48 (s, 4H); 13C NMR (101 MHz, METHANOL-d4) d 135.6, 131.9, 129.2, 95.6, 86.6, 53.9; ESI-MS: C12H1102+ [M+H]+, 187.1; found 187.1. 33: 1,2-Phenylene)dipropiolonitrile.
The compound was obtained as the only product of the standard Mn02 oxidation protocol. Reaction time: 75 minutes. Brown solid, yield: 42%. 1H NMR (400 MHz, OL-d4) 8 7.89 (dd, J: 3.30, 5.80 Hz, 2H), 7.73 (dd, 12 3.30, 5.80 Hz, 2H); 13C NMR (101 MHz, METHANOL—d4) 8 136.0, 133.5, 126.5, 105.5, 80.2, 67.2; GC-ESI-MS: C12H5N2+ [M+H]+, 177.0; found 177.0. 3,3'-(1,3-Phenylene)dipropiolonitrile (34): propargylic alcohol Cul PcICI2P(Ph) MnOz NHa DIPEA DMF MgSO4 IPA THF 34a: 3,3'-(1,3-Phenylene)bis(propyn-l-ol).
Same procedure as for the synthesis of 33a. Brownish solid, yield: 55%. 1H NMR (400 MHz, METHANOL—d4) 8 7.47 (s, 1H), 7.36 - 7.43 (m, 2H), 7.29 - 7.36 (m, 1H), 4.41 (s, 4H); 13C NMR (101 MHz, METHANOL—d4) 8 135.3, 132.5, 129.8, 124.8, 89.7, 84.5, 51.2; ESI-MS: C12H1102+ [M+H]+, 187.1; found 187.0. 34: 3,3'-(1,3-Phenylene)dipropiolonitrile.
The compound was obtained as the only product of the standard MnOz oxidation protocol. Reaction time: 2 hours. Brown solid, yield: 35%.
W0 20151001117 50 1H NMR (400 MHz, METHANOL-d4) 5 8.10 (d, J = 1.50 Hz, 1H), 7.93 (dd, J = 1.50, 8.00 Hz, 1H), 7.63 (t, J: 8.00 Hz, 2H); 13C NMR (101 MHz, METHANOL-d4) 5 139.3, 137.8, 131.2, 120.0, 105.7, 81.7, 64.2; GC-ESI-MS: ClesNz+ [M+H]+, 1770, found 177.1. 3,3'-(1,4-Phenylene)dipropiolonitrile (35): Br gylic alcohol Cul PdCI2(PPh3)2 MnOZ NH3 DIPEA, DMF MgSO4 IPA, THF 353: 3,3'-(1,4-Phenylene)bis(prop-2—ynol).
Sarne procedure as for the synthesis of 333, but refluxed for 72 hours. Brownish solid, yield: 35%. 1H NMR (400 MHz, METHANOL-d4) 8 7.39 (s, 4H), 4.41 (s, 4H); 13C NMR (101 MHz, METHANOL-d4) 5 132.6, 124.3, 101.4, 90.8, 84.9, 51.2; ESI-MS: C12H1102+ [M+H]+, 187.1; found 187.1. : 3,3'-(1,4-Phenylene)dipropiolonitrile.
The compound was ed as the only product of the standard MnOz oxidation protocol. on time: 2 hours. Brown solid, yield: 19%. 1H NMR (400 MHz, METHANOL-d4) 6 7.94 (s, 4H); 13C NMR (101 MHz, METHANOL—d4) 8 135.0, 121.6, 105.5, 82.0, 65.9; GC-ESI-MS: C12H5N2+ [M+H]+, 177.0; found 177.0.
Cornpounds 33-35 can be used for rebridging ) according to the invention. tert-butyl (S)((tert-butoxycarbonyl)amino)(4- (((triflu0r0methyl)sulfonyl)0xy)phenyl)pr0panoate (36b): WO 20151001117 51 OH O\S//O F d’XF >L O F CAN >L JOL 07< o H H Oj< o 0 To a cooled to 0 °C solution of tert-butyl 2-{[(tert-butoxy)carbony1]amino}(4- hydroxypheny1)propanoate (1 eq., 518 mg, 1.54 mmol) in pyridine (2.5 mL), triflic ide (1.1 eq., 476 mg, 0.28 mL, 1.69 mmol) was added dropwise over 20 minutes (using syringe presser). The resulting dark solution was let to warm up to room temperature, poured into water (10 mL), and extracted with ethyl ester (15 mL). The ether t was washed sequentially with water (5 mL), 1N HCl (2x5 mL), water (5 mL), brine (5 mL), dried over MgSO4, and evaporated to give the targeted product (614 mg, 1.31 mmol, 85 %) as a dark-red oil. The product was used in the next step without fiarther purification. 1H NMR (400 MHZ, FORM-d) 8 7.03 - 7.18 (m, 3H), 6.82 - 7.03 (m, 2H), 4.87 (d, J = 7.28 Hz, 1H), 4.24 (d, J: 7.03 Hz, 1H), 2.65 - 2.95 (m, 2H), 1.17 (s, 9H), 1.21 (s, 9H). 13C NMR (101 MHz, CHLOROFORM-d) 8 170.5, 148.5, 137.3, 131.3, 121.1, 120.3, 117.1, 82.5, 80.0, 54.7, 38.1, 28.3, 27.9. tert-butyl (S)((tert-but0xycarb0nyl)amin0)(4-(3-hydr0xypr0pyn yl)phenyl)pr0pan0ate (36a): O\S/§D F o" f.
XOAHO —. #0 O)L OW< H OK O 0 36b 36a To a solution of phenoltryphlate (1 eq., 136 mg, 0.291 mmol) in morpholine (1 mL) were consequently added PdC12(PPh3)2 (5 %, 10.2 mg, 0.0145 mmol), CuI (10 %, .53 mg, 0.0291 mmol), and propargylic alcohol (2 eq., 32.6 mg, 0.0343 mL, 0.581 W0 20151001117 52 mmol). The obtained reaction mixture was degassed and heated at 60 0C for 24 hours.
The resulting black solution was poured into water (10 mL), extracted with EtOAc (3X10 mL). The united organic layers were washed with 1N HCl (2X10 mL), water (1X10 mL), dried over MgSO4 and evaporated to give crude product, which after purification by flash chromatography gave the targeted product (8.73 mg, 0.0232 mmol, 8 %) as a yellowish solid. 1H NMR (400 MHz, METHANOL-d4) 5 7.30 = 7.78 Hz, 2H), 7.14 - 7.45 (m, J - 7.30 (m, J = 8.03 Hz, 2H), 4.40 (s, 2H), 4.18 = 6.27, 13.80 Hz, - 4.32 (m, 1H), 3.06 (dd, J 1H), 2.91 (dd, J: 8.66, 13.68 Hz, 1H), 1.45 - 1.53 (m, 1H), 1.42 (d, J: 3.26 Hz, 19H). utyl rt-butoxycarbonyl)amin0)(4-(cyanoethynyl)phenyl)pr0pan0ate (36): O 0 OX OX HN o —’ “N 0 HO é Y 7 Y 0% N’ 0% 36a 36 Product was synthesized according to general procedure of MnOz-oxidation. on time: 2 hours; yield: 56%. 1H NMR (400MHz, CHLOROFORM-d) 8 = 7.54 (d, J = 8.2 Hz, 2H), 7.24 (d, J: 8.2 Hz, 2H), 5.05 (d, J: 7.3 Hz, 1H), 4.46 (td, J: 6.1, 7.3 Hz, 1H), 3.14 (dd, J: 6.1, 13.7 Hz, 1H), 3.05 (dd, J: 6.1, 13.7 Hz, 1H), 1.42 (s, 9H), 1.41 (s, 9H). 13C NMR (101MHz, CHLOROFORM-d) 8 = 170.3, 154.9, 141.4, 133.4, 130.1, 115.9, 105.5, 82.9, 82.5, 79.9, 63.2, 54.5, 38.8, 28.3, 27.9.
Compound 36 can be used for purification and/or immobilization according to the invention. 4-(cyan0ethynyl)-N-(2-(2-(2-(5-((3aS,4S,6aR)0x0hexahydr0-1H-thien0[3,4- d]imidaz0lyl)pentanamido)ethoxy)ethoxy)ethyl)benzamide (3 7) WO 20151001117 53 HTNH H 0 “\\/\)J\N/\/O\/\O/\/H H8 0 To the solution of N—(2-(2-(2-aminoethoxy)eth0xy)ethyl)((3aS,4S,6aR) 0x0hexahydr0—1H-thien0[3,4—d]imidazolyl)pentanamide (1 eq., 222 mg, 0.593 mmol) in dry DMF (1 mL) was added sodium 4-((4-(cyanoethynyl)benz0yl)0xy)-2,3,5,6- tetrafluorobenzenesubconate (1.2 eq., 300 mg, 0.712 mmol) and DIEA (5.] eq., 39] mg, 0.5 mL, 3.03 mmol). The mixture was stirred at r.t. for 3 hours and then purified by semi-preparative HPLC to give the desired product (68.8 mg, 0.13 mmol, 22% yield) as a yellow oil. 1H NMR z ,METHANOL-d4) 5 = 7.92 (d, J = 8.5 Hz, 2 H), 7.81 (d, J: 8.5 Hz, 2 H), 4.49 (dd, J = 4.8, 7.8 Hz, 1 H), 4.30 (dd, J: 4.5, 7.8 Hz, 1 H), 3.71 — 3.56 (m, 8 H), 3.54 (t, J: 5.5 Hz, 2 H), 3.34 (t, J: 5.5 Hz, 2 H), 3.24 - 3.14 (m, 1 H), 2.92 (dd, J: 4.8, 12.8 Hz, 1 H), 2.70 (d, J: 12.8 Hz, 1 H), 2.19 (t, J: 7.4 Hz, 2 H), 1.78 — 1.50 (m, 4 H), 1.48 - 1.35 (m, 2 H). 13C NMR (101MHz ,METHANOL-d4) 5 = 176.3, 168.8, 166.2, 138.9, 135.0, 129.1, 121.5, 105.9, 83.1, 71.5, 71.4, 70.7, 70.6, 64.6, 63.5, 61.8, 57.1, 41.2, 40.4, 36.9, 29.9, 29.6, 27.0.
W0 01117 54 ((4-(Cyanoethynyl)phenyl)amino)oxobutyl)-amino) oxoethyl)tris(2,4,6-trimethoxyphenyl)phosphonium trifluoroacetate (38): NH2 0 propargylic alcohol. OWNHBOC Boo GABA- WNHBOCPd(PPh3)2C|2 —,HN _, EDC, TEA, DMAP DIPEA DCM, 0°C THF, 25°C /0 J, O MnOZV 3‘ NH3(|PA)V 3“ 0‘ M91304, P 0’ THF O O BocHN _ \ TMPP-Ac—OSu O NH 38 38a 38b 38d: tert-Butyl (4-((4-iodophenyl)amino)-4—oxobutyl)carba-mate.
To the cooled to 0°C solution of Boc-GABA (1 eq., 0.928 g, 4.57 mmol), TEA (3 eq., 1.39 g, 1.9 mL, 13.7 mmol) and DMAP (0.05 eq., 0.0279 g, 0.228 mmol) in DCM (11.7 mL), EDC (1 eq., 0.875 g, 4.57 mmol) was added. The obtained reaction mass was stirred for another 10 minutes at 0°C, an ice bath was removed, and p-iodoaniline (1 eq., 1 g, 4.57 mmol) was added and the reaction was left overnight at 25°C. The ed reaction mass was washed with 1M HCl (2x20 rnL), water (1X20 mL), and dried over Na2S04 to give 38d (1125 mg, 2.79 mmol, 61 %), which was used without fiarther purification. 1H NMR (400 MHz, CHLOROFORM-d) 8 9.04 (br. s., 1H), 7.58 = 8.50 — 7.72 (m, J Hz, 2H), 7.37 = 8.50 Hz, 2H), 4.81 - 7.51 (m, J (br. s., 1H), 3.27 (rn, 2H), 2.30 - 2.50 (m, 2H), 1.88 (m, 2H), 1.49 (s, 9H); 13C NMR (101 MHz, CHLOROFORM—d) 8 174.2, 157.4, 137.8, 120.9, 120.0, 87.3, 77.0, 33.1, 32.8, 28.4, 26.0; ESI-MS: C15H22N203+ [M+H]+, 405.0; found 405.1.
WO 2015001117 55 38c: tert-Butyl (4-((4-(3-hydroxypropynyl)phenyl)-amino) oxobutyl)carbamate.
Synthesised following the protocol B for shira coupling. Yellowish solid, yield: 79%. 1H NMR (400 MHz, METHANOL-d4) 5 7.54 = 8.50 Hz, 2H), 7.34 - 7.58 (m, J - 7.38 (m, J = 8.50 Hz, 2H), 4.40 (s, 2H), 3.13 (t, J: 6.90 Hz, 2H), 2.41 (t, J: 740 Hz, 2H), 1.81-1.89 (m, 2H), 1.44 (s, 9H); 13C NMR (101 MHz, METHANOL—d4) 8 174.0, 158.6, 140.1, 133.2, 120.8, 119.5, 88.3, 85.3, 80.1, 51.3, 40.9, 35.3, 28.8, 27.1; ESI-MS: C18H25N204+ [M+H]+, 332.1; found 332.0. 38b: tert-Butyl (4-((4-(cyanoethynyl)phenyl)amino)0X0butyl)carbamate.
Synthesised using standard protocol of Mn02 oxidation. Reaction time: 1 hour. White solid, yield: 85%. 1H NMR (400 MHz, METHANOL—d4) 5 7.61 = 8.80 Hz, 2H), 7.54 - 7.65 (m, J - 7.59 (m, J: 8.50 Hz, 2H), 3.04 (t, J: 6.85 Hz, 2H), 2.34 (t, J: 7.40 Hz, 2H), .81 (m, 2H), 1.34 (s, 9H); 13C NMR (101 MHz, METHANOL-d4) 8 174.3, 159.1, 143.8, 135.7, 120.8, 112.8, 106.3, 84.6, 62.7, 40.8, 35.3, 34.8, 28.8, 26.9; ESI—MS: C18H22N303+ [M+H]+, 328.1; found 328.1. 383: 4-((4-(Cyanoethynyl)phenyl)amino)oxobutanaminium trifluoroacetate.
To a suspension of 38b (1 eq., 62.8 mg, 0.192 mmol) in DCM (1 mL), TFA (20 eq., 285 uL, 3.83 mmol) was added and the obtained solution was stirred at 25°C for 30 minutes.
The target product 383 (TFA salt, 65.0 mg, 0.19 mmol, 99 %) was obtained after the evaporation of the reaction mass and was used without further purification in the next step. 1H NMR (400 MHz, METHANOL-d4) 5 7.71 = 9.15 Hz, 2H), 7.63 - 7.79 (m, J - 7.70 (m, J = 9.15 Hz, 2H), 3.04 (t, J= 6.80 Hz, 2H), 2.6 (t, J= 7.05 Hz, 2H), 1.98-2.08 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 173.1, 143.6, 135.7, 120.7, 112.9, 106.2, 84.5, 62.7, 40.4, 34.5, 24.0; : C13H14N30+ [M+H]+, 228.1; found 228.1. 38: (2-((4-((4-(Cyanoethynyl)phenyl)amino)0X0butyl)amino) oxoethyl)tris(2,4,6-trimethoxyphenyl)phosphonium trifluoroacetate.
To the solution of 383 (1 eq., 10.1 mg, 0.0296 mmol) in DMF (250 uL), TEA (1 eq., 4 uL, 0.0296 mmol) was added. TMPP-Ac-OSu (1 eq., 22.7 mg, 0.0296 mmol) was added to the ed solution and the reaction mass was for 15 minutes at room temperature.
W0 20151001117 56 The crude product was purified by HPLC to isolate 38 (9.9 mg, 0.0126 mmol, 42 %) as a main product. 1H NMR (400 MHz, METHANOL-d4) 5 7.44 - 7.59 (m, 4H), 6.13 (d, J: 4.52 Hz, 6H), 3.75 (s, 9H), 3.50 (s, 18H), 3.00 (td, J= 7.91, 15.31 Hz, 2H), 2.26 (t, J: 6.90 Hz, 2H), 1.64-1.75 (m, 2H), 1.25-1.43 (m, 2H); 13C NMR (101 MHz, METHANOL—d4) 5174.2, 167.4, 167.4, 165.3, 143.6, 135.8, 120.6, 112.8, 106.3, 92.2 (d, J: 8 Hz), 84.5, 62.7, 56.5, 56.2, 37.5, 29.4 (d, J: 64 Hz), 27.9, 27.7, 24.9; ESI-HRMS: N3011P+ [M]+, 800.29427; found 800.29401. (5-((4-(Cyanoethynyl)phenyl)amino)oxopentyl)tris(2,4,6- hoxyphenyl)phosphonium bromide (39): 0 Br lowBr gylic YWalcohol, HN Pd(PPh3)2C|2 —’ MOI —’ DIPEA DIPEA \ DCM THF \ 905$ MnOz‘ NH3 (IPA), M9304, O/flij—YN OWE I N\©\ .flToluene, 60°C\\N 39 HN\<j:a\\\N 39d: 5-Bromopentanoyl chloride.
Degassed solution of 5-brornopentanoic acid (1 eq., 2.85 g, 15.7 mmol) and SOC12 (1 eq., 1.87 g, 1.14 mL, 15.7 mmol) in DCM (50 mL) was refluxed for 3 hours. The obtained reaction mass was evaporated under d pressure to give 39d (3.11 g, 100%) as a yellowish oil. The crude product was used in the next step without purification. 39c: S-Bromo-N-(4-iodophenyl)pentanamide.
Solution of 39d (1 eq., 3.11 g, 15.7 mmol) in DCM (50 mL) was poured into a cooled to -78°C solution of 4-iodoaniline (1 eq., 3.45 g, 15.7 nlrnol) and DIPEA (1 eq., 2.03 g, 2.6 WO 2015001117 57 mL, 15.7 mmol) in DCM (50 mL). Obtained on mass was allowed to warm to room ature, stirred for another 30 min, washed with 1N HCl (2X25 mL), water (1x25 mL), dried over Na2S04 and evaporated to give 39c (5.60 g, 14.66 mmol, 93%) as brown solid. 1H NMR (400 MHz, METHANOL-d4) 5 7.62 - 7.66 (m, 2H), 7.38 — 7.43 (m, 2H), 3.50 (t, J= 6.53 Hz, 2H), 2.42 (t, J: 7.28 Hz, 2H), 1.81 - 1.98 (m, 4H), 1.37 - 1.42 (m, 1H); 13C NMR (101 MHz, METHANOL—d4) 5 174.0, 140.5, 138.9, 123.1, 87.6, 36.9, 33.8, 33.4, 25.3; : C11H14BrINO+ [M+H]+, 381.9; found 381.8. 39b: S-Bromo-N-(4-(3-hydroxyprop-l-ynyl)phenyl)pentanamide: sised following the ol A for Sonogashira coupling. Brown solid, yield: 92%. 1H NMR (400 MHz, METHANOL-d4) 57.49 = 8.53 Hz, 2H), 7.32 - 7.63 (m, J - 7.43 (m, J = 8.53 Hz, 2H), 4.40 (s, 2H), 4.26 (s, 1H), 3.50 (t, J = 6.53 Hz, 2H), 2.43 (t, J = 7.15 Hz, 2H), 1.90 - 2.04 (m, 2H), 1.74 - 1.90 (m, 2H), 1.32 (s, 1H); 13C NMR (101 MHz, METHANOL-d4) 5173.9, 140.0, 133.1, 120.7, 119.6, 88.2, 85.2, 51.2, 36.8, 33.7, 33.3, 25.3; ESI—MS: C14H17BrNO+ [M+H]+, 3100; found 310.0. 393: 5-Brom0-N-(4-(cyanoethynyl)phenyl)pentanamide. 2M solution of NH3 (4 eq., 94.8 mg, 5.56 mmol) in IPA and anhydrous MgSO4 (15 eq., 2511 mg, 20.9 mmol) were added to a stirred solution of 39b (1 eq., 431 mg, 1.39 mmol) in THF (3.42 mL). Activated MnOz (15 eq., 1814 mg, 20.9 mmol) was added to the solution and the resulting mixture was stirred at room temperature for 4 hours (controlled by TLC, no more starting alcohol; NB: too long on time gives hydrolysis product), diluted with DCM (13 mL). The mixture was filtered, washed thoroughly with DCM and the combined filtrates were concentrated under reduced pressure. The solid residue was purified by flash chromatography (EtOAc-cyclohexane, min gradient from 0 to 100% of EtOAc) to give 39 as a white solid (288 mg, 0.946 mmol, 68 %). 1H NMR (400 MHz, METHANOL-d4) 57.69 = 8.78 Hz, 2H), 7.59 - 7.79 (m, J - 7.69 (m, J = 8.78 Hz, 2H), 3.50 (t, J = 6.53 Hz, 2H), 1.79 - 1.99 (m, 4H), 1.26 (t, J = 7.15 Hz, 2H); 13C NMR (101 MHz, METHANOL-d4) 5174.3, 143.8, 135.7, 120.7, 112.8, 106.2, 101.4, 84.6, 37.0, 33.7, 33.4, 25.2. ESI-MS: C14H14BrN20+ [M+H]+, 304.0; found 304.0.
W0 20151001117 5 8 39: (5-((4-(Cyanoethynyl)phenyl)amino)0X0pentyl)tris(2,4,6-trimethoxy— phenyl)phosphonium bromide. 39a (1 eq., 20 mg, 0.0655 mmol) and tris(2,4,6-trimethoxy—phenyl)phosphane (TMPP, 1.2 eq., 41.9 mg, 0.0786 mmol) were dissolved in dry toluene (1 mL) and stirred overnight at room temperature. 39 (TFA salt, 22 mg, 39%) was obtained after reversephase HPLC as a white solid. 1H NMR (400 MHz, METHANOL-d4) 8 7.51—7.55 (m, 4H), 6.13 (d, J = 4.77 Hz, 6H), 3.75 (s, 9H), 3.50 (s, 18H), 3.00 (td, J= 6.90, 15.31 Hz, 2H), 2.26 (t, J: 6.90 Hz, 2H), 1.73 (m, 2H), 1.22 - 1.45 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 174.2, 167.4, 165.3, 143.6, 135.8, 120.6, 112.8, 106.2, 93.6, 92.3, 92.2, 84.5, 62.7, 56.3, 37.5, 29.7, 27.7, 24.9; ESI-HRMS: C41H46N2010P+ [M]+, 846; found 757.29552. (4-(4-(Cyanoethynyl)benzamido)butyl)tris(2,4,6-trimethoxyphenyl)phosphonium trifluoroacetate (40): 1. SOCI2 ,A . HN/\/\Br 2. propargyllc HN/V\Br alcohol, HO NH2 Br Pd(PPh3)2C|2 O | —> —> DIPEA DIPEA o \ DCM, 0°C THF, 25°C \ 406 401) CH MnOZY NH3 (IPA), 0 g M9504, I Z:TF THF flP\—\O_;O HNMBr TMPP o Toluene 60°C \\\\ \\ 40c: N-(4-Bromobutyl)iodobenzamide. 4-iodobenzoic acid (1 eq., 1.45 g, 5.85 mmol) was heated at 110 °C in SOClz (9 eq., 3.8 mL, 52.6 mmol) until complete dissolving (around 15 min). Excess of SOClz was removed in vacuo and obtained solid was poured into DCM (15 mL), cooled to -78 °C and DIPEA (3.1 eq., 3 mL, 18.2 mmol) was added under vigorous stirring. 3- ropylamine hydrobromide (1.5 eq., 1.90 g, 8.77 mmol) was added to the obtained on mass was left stirring for 5 minutes still at -78 °C, let to warm up to room temperature, while stirring for r 20 minutes. Ethyl acetate (100 mL) was W0 01117 59 added with 1M HCl (5 mL), obtained solid was filtered (product), washed with water and dried to yield 40c (2.09 g, 5.67 mmol, 97 %) as a white solid. 1H NMR (400 MHz, METHANOL-d4) 5 7.85 (m, J = 8.40 Hz, 2H), 7.58 (m, J = 8.40 Hz, 2H), 3.48-3.56 (m, 4H), 2.12-2.23 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 160.1, 139.2, 136.0, 132.8, 102.4, 50.1, 43.2, 23.0; ESI-MS: C10H12BrINO+[M+H]+, 367.9; found 368.0. 40b: N-(4-Bromobutyl)(3-hydroxypropynyl)benzamide.
Synthesised following the protocol B for Sonogashira coupling. Brown solid, yield: 81%. 1H NMR (400 MHz, METHANOL-d4) 5 7.73 (m, J = 8.40 Hz, 2H), 7.38 (m, J = 8.40 Hz, 2H), 4.37 (t, J = 5.30 Hz, 2H), 4.34 (s, 2H), 3.51 (t, J= 5.80 Hz, 2H), 1.92-1.98 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 159.2, 134.2, 132.4, 128.2, 127.1, 91.2, 84.8, 67.2, 51.2, 43.3, 22.5; ESI-MS: C13H15BTNOZ+[M+H]+, 295.0; found 295.0. 403: N-(3-Bromopropyl)(cyanoethynyl)benzamide.
Synthesised using standard protocol of MnOz oxidation. Reaction time: 45 minutes.
Brown solid, yield: 52%. 1H NMR (400 MHz, METHANOL-d4) 5 7.90 (m, J = 8.50 Hz, 2H), 7.80 (m, J = 8.50 Hz, 2H), 3.42-3.55 (m, 2H), .35 (m, 2H), .23 (m, 2H), 1.92-1.98 (m, 2H); 13C NMR (101 MHz, OL-d4) 5 168.7, 138.8, 134.9, 128.9, 121.4, 105.8, 83.0, 67.3, 41.9, 39.8, 22.8; ESI-MS: C13H12BrN20+ [M+H]+, 291.0; found 291.2. 40: (4-(4-(Cyanoethynyl)benzamido)butyl)tris(2,4,6- trimethoxyphenyl)phosphonium trifluoroacetate. 403 (1 eq., 30 mg, 0.103 mmol) and tris(2,4,6-trimethoxyphenyl)phosphane (TMPP, 1 eq., 54.9 mg, 0.103 mmol) were dissolved in dry toluene (2 mL). The obtained solution was left overnight at room temperature. The precipitate was filtered, resolubilised in DMSO, and purified by HPLC to give 40 (35 mg, 0.0409 mmol, 40 %) as a white solid. 1H NMR (400 MHz, METHANOL-d4) 57.75 (d, J= 8.50 Hz, 2H), 7.69 (d, J = 8.50 Hz, 2H), 6.16 (d, J = 4.70 Hz, 2H), 3.76 (s, 9H), 3.51 (s, 18H), 3.35 (t, J = 7.10 Hz, 2H), 2.98-3.10 (m, 2H), 1.53-1.64 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 5 168.5, 167.5, 165.3, 138.7, 134.9, 128.8, 121.2, 105.8, 94.0, 92.9, 92.3, 82.9, 64.5, 56.5, 41.7, 27.8, 25.7; ESI-HRMS: C40H44N2010P+ [M]+, 743.22728; found 743.23946.
WO 2015001117 60 Compounds 38-40 can be used for detection and/or separation method according to the invention. 3-(4-(2,5-Dioxo—2,5-dihydro-1H-pyrrolyl)phe-nyl)propiolonitrile (41): o 0&0_ NH2 HN Maleic anhydride TfZO, K2C03 —> —> acetone DMF I I I I I I N INI INI 7 41a 41 413: ((4-(Cyanoethynyl)phenyl)amino)0X0butenoic acid.
To the solution of 7 (1 eq., 76.8 mg, 0.541 mmol) in acetone (2 mL), maleic anhydride (2 eq., 106 mg, 1.08 mmol) was added. A yellowish solid was obtained after about 7 hours of stirring. The reaction mass was evaporated, an excess of maleic anhydride and maleic acid was washed with methanol. 413 (127 mg, 0.53 mmol, 98 %) was ed as yellowish solid, no further purification was needed. 1H NMR (400 MHz, DMSO-ds) 8 12.90 (br. s., 1H), 10.70 (s, 1H), 7.62 - 7.90 (m, 4H), 6.50 (d, J: 11.90 Hz, 1H), 6.34 (d, J: 11.90 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) 8 166.8, 163.8, 142.4, 135.0, 131.7, 130.1, 119.3, 110.2, 105.6, 84.3, 61.9; ESI-MS: C13H7N203' [M-H]', 239.0; found 239.0. 41: 3-(4-(2,5-Dioxo-2,5-dihydro-IH-pyrrolyl)phenyl)-propiolonitrile.
To the solution of 413 (1 eq., 75 mg, 0.312 mmol) in dry DMF (1.21 mL) trifluoroacetic anhydride (2 eq., 86.9 uL, 0.624 mmol) was added. Stirring continued for r 5 minutes at room temperature and K2C03 (3 eq., 129 mg, 0.937 mmol) was added. The reaction mass stirred for another 60 s, then directly purified by HPLC to give 41 (65.9 mg, 0.297 mmol, 95%) as a slightly yellow solid. 1H NMR (400 MHz, DMSO-ds) 5 7.81 (m, J: 8.50 Hz, 2H), 7.52 (m, J: 8.50 Hz, 2H), 6.96 (s, 2H); 13C NMR(101 MHz, DMSO-d6) 8169.0, 134.4, 134.0, 126.0, 117.3, 117.0, 82.2, 78.5, 62.3; ESI-MS: C13H7N202+ [M+H]+, 2230; found 229.9. nd 41 can be used for a bioconjugation method according to the invention.
WO 2015001117 61 2,5-Dioxopyrrolidinyl 5-((4-(cyanoethynyl)-phenyl)-amino)0X0pentanoate (42) : 0 O OH I? NH2 flo O NH O NH Glutaric anhydride DCC, NHS —> _> acetone TEA, DCM N INI INI 42a 42 42a: 5-((4-(Cyanoethynyl)phenyl)amino)0X0pentanoic acid.
To a solution of 7 (1 eq., 200 mg, 1.41 mmol) in acetone (1 mL), ic anhydride (2 eq., 321 mg, 2.81 mmol) was added. The obtained solution was stirred for 24 hours at room temperature. Acetone was evaporated, the crude product was recrystallised from IPA-cyclohexane to give 423 (324 mg, 1.27 mmol, 90 %) as a grey solid. 1H NMR (400 MHz, s) 810.21 (s, 1H), 8.15 (br. s., 1H), 7.60 (d, J = 8.72 Hz, 2H), 7.52 (d, J = 8.72 Hz, 2H), 2.52-2.62 (m, 4H), 2.22-2.32 (m, 2H); 13C NMR (101 MHz, DMSO-d6) , 168.5, 140.9, 134.2, 119.0, 111.9, 105.4, 84.1, 63.3, 30.1, 29.0, 21.2; ESI-MS: C14H11N203' [M-H]', 255.1; found 255.1. 42: 2,5-Dioxopyrrolidinyl 5-((4-(cyanoethynyl)phenyl)-amino)oxopentanoate.
To a solution of 42a (1 eq., 18 mg, 0.0702 mmol) in DCM (1 mL), DCC (1.02 eq., 14.8 mg, 0.0716 mmol) and TEA (1 eq., 6.52 mg, 0.00895 mL, 0.0644 mmol) were added.
The obtained reaction mass was d for 5 minutes, NHS (1 eq., 8.08 mg, 0.0702 mmol) was added. The resulting solution stirred for r 2 hours at room temperature. The crude product was purified by flash chromatography (cyclohexane- EtOAc) to give 42 (6.45 mg, 0.0183 mmol, 26%) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d) 8 8.27 (br. s., 1H), 7.64 (d, J = 8.78 Hz, 2H), 7.57 (d, J = 8.78 Hz, 2H), 2.94 (s, 4H), 2.74 (t, J = 6.53 Hz, 2H), 2.52 (t, J = 6.90 Hz, 2H), 2.23 (m, 2H); 13C NMR (101 MHz, CHLOROFORM-d) d 170.3, 169.5, 168.2, 141.3, 134.6, 119.4, 112.4, 105.7, 83.2, 62.9, 35.6, 29.9, 25.7, 21.2; ESI—MS: C18H16N305+ [M+H]+, 353.1; found 353.2.
Compound 42 can be used for a bioconjugation method according to the invention.
WO 2015001117 62 3-(4-azidophenyl)propiolonitrile (43): HZN : _N |AN,TMSN3 N :N —————> ,3 K2C03'ACN _// 7 (1 eq., 151 mg, 1.07 mmol) was dissolved in acetonitrile (2.34 mL) in a 25 mL roundbottomed flask and cooled to 0°C in an ice bath. To this stirred mixture was added isoamyl e (IAN, 1.5 eq., 215 uL, 1.6 mmol) followed by trimethylsilyl azide (1.2 eq., 147 mg, 0.168 mL, 1.28 mmol) dropwise. The resulting solution was stirred at room temperature for 45 minutes. The reaction mixture was trated under vacuum and the crude product was resolubilised in EtOAc, washed with water, dried and evaporated to give 43 (177 mg, 1.06 mmol, 99%). 1H NMR (400 MHz, ACETONITRILE-ds) 6 7.58 = 8.78 Hz, 2H), 7.11 — 7.81 (m, J - 7.26 (m, J = 8.78 Hz, 2H); 13C NMR (101 MHz, ACETONITRILE-ds) 5 144.9, 136.0, 120.4, 113.6, 105.9, 83.4, 62.9; GC-ESI-MS: C9H5N4+ [M+H]+, 169.0; found 169.0.
S-Azido-N-(4-(cyanoethynyl)phenyl)pentanamide (44): NH2 HN CIJ‘J\/\/\N3 44a N3 ll TEA,DCM 44a: opentanoyl chloride. -azidopentanoic acid (1 eq., 1.1 g, 6.99 mmol) was refluxed in SOClz (10 eq., 5.1 mL, 69.9 mmol) for 30 minutes. Excess of SOClz was d in vacuo and the obtained crude solid was used in the next step without purification. 44: S-Azido-N-(4-(cyanoethynyl)phenyl)pentanamide. 7 (1 eq., 16.1 mg, 0.113 mmol) and TEA (1.5 eq., 24 uL, 0.17 mmol) were dissolved in DCM (3 mL), cooled to -78 °C, and 443 (1.1 eq., 20.1 mg, 0.125 mmol) was added to the reaction e that was then left to warm to room temperature while stirring for another 1 hour. The reaction mass was washed with 1M HCl (2x1 mL), water (2 mL), WO 2015001117 63 dried over Na2S04, and evaporated to give crude product, which was purified by flash chromatography to give 44 (25.5 mg, 0.101 mmol, 89 %) as a grey solid. 1H NMR (400 MHz, METHANOL-d4) 8 7.58 = 8.70 Hz, 2H), 7.40 - 7.67 (m, J - 7.58 (m, J: 8.70 Hz, 2H), 3.23 - 3.28 (m, 2H), 2.34 (t, J: 7.28 Hz, 2H), 1.61 - 1.72 (m, 2H), 1.49 - 1.61 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8173.0, 144.0, 135.7, 134.3, 120.7, 113.0, 106.2, 84.5, 40.4, 34.4, 28.8, 24.0; ESI-MS: C14H14NsO+ [M+H]+, 2681; found 268.1. 1-[4-(Cyanoethynyl)benzyl]-3,3,6,6-tetramethyl-4,5-didehydr0-2,3,6,7- tetrahydrothiepinium triflate (45): propargylic alcoholCul PdC'z(PPha) MnOz NHa NBS DIPEA, DMF \\ \ —’ MgSO4 IPA THF \ MW,CC|4 ‘OTf BI’ I l 8+ TMTH LiOTf, DCM/HZO \\ \\ N \\ 45a 45 45c: 3-(p-Tolyl)prop-2—ynol.
Synthesised using protocol A for Sonogashira coupling. Yellowish solid, yield: 88%. 1H NMR (400 MHz, ITRILE-ds) 6 7.25 = 8.03 Hz, 2H), 7.04 - 7.49 (m, J - 7.25 (m, J = 8.03 Hz, 2H), 4.34 (d, J: 6.02 Hz, 2H), 3.31 (t, J = 6.02 Hz, 1H), 2.32 (s, 3H); 13C NMR (101 MHz, ACETONITRILE-dg) 8139.8, 132.4, 130.3, 120.8, 88.8, 85.1, 51.2, 21.5; ESI-MS: 0+ [M+H]+, 146.1; found 146.0. 45b: 3-(p-Tolyl)propiolonitrile.
The compound was obtained as the only t of the standard MnOz oxidation protocol. Reaction time: 3 hours. White solid, yield: 67%. 1H NMR (400 MHz, METHANOL-d4) 87.37 = 8.03 Hz, 2H), 7.02 - 7.59 (m, J - 7.31 (m, J= 8.03 Hz, 2H), 2.29 (s, 3H); 13C NMR (101 MHz, METHANOL-d4) 143.2, WO 2015001117 64 133.3, 129.5, 114.0, 104.8, 83.2, 61.3, 20.4; ESI-MS: C10H8N+[M+H]+, 141.1; found 141.0. 453: 3-(4-(Bromomethyl)phenyl)propiolonitrile. d solution of 45b (1 eq., 68 mg, 0.482 mmol) in DCM (1 mL) was MW- irradiated (100°C) for 5 minutes. The reaction mixture was evaporated, the crude was purified by preparative HPLC to give 453 (42.4 mg, 0.193 mmol, 40 %) as a yellowish solid. 1H NMR (400 MHz, CHLOROFORM-d) 5 7.56 - 7.71 (m, J = 8.28 Hz, 2H), 7.40 - 7.49 (m, J = 8.28 Hz, 2H), 4.48 (s, 2H); 13C NMR (101 MHz, CHLOROFORM-d) 141.8, 133.9, 129.5, 117.5, 105.3, 82.3, 63.7, 31.8; GC-ESI-MS: C10H7BrN+ , 219.0; found 219.0. 45: 1-[4-(Cyanoethynyl)benzyl]-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7- tetrahydrothiepinium e.
To a degassed solution of 453 (1 eq., 43.7 mg, 0.199 mmol) and TMTH (1.29 eq., 43 mg, 0.255 mmol; synthesised following previously described procedures“) in DCM (1.34 mL), a solution of LiOTf (11.6 eq., 360 mg, 2.31 mmol) in distilled and degased H20 (0.668 mL) was added. The obtained biphasic mixture was vigorously stirred for days at 25 °C (degasing once per day). Two phases were separated, the organic one was washed with DCM (5X2 mL). United organic ons were evaporated and the crude was purified by HPLC to give 45 (46.9 mg, 0.111 mmol, 56 %) as colourless oil (crystallizes slowly at 0 °C to yield a white solid). 1H NMR (400 MHz, CHLOROFORM-d) 57.65 = 8.03 Hz, 2H), 7.56 - 7.73 (m, J - 7.65 (m, J = 8.03 Hz, 2H), 5.07 (s, 2H), 4.12 (d, J = 12.30 Hz, 2H), 3.72 (d, J = 12.30 Hz, 2H), 1.36 (s, 6H), 1.30 (s, 6H); 13C NMR (101 MHz, DMSO-d6) 8135.1, 133.6, 131.9, 117.8, 106.4, 105.8, 83.3, 63.6, 60.1, 43.2, 34.6, 26.4, 25.4; HR-ESI-MS: C20H22NS+ [M]+, 308.1; found 308.1.
Compounds 43-45 can be used for a click chemistry (such as reaction click - azide) according to the invention. nd 45 can be used for strain promoted click ing to the invention.
W0 01117 65 1 - ({4 - [1 - {[2 - ({3 - Carboxylato [6 - (dimethylamino) (dimethyliminiumyl) - 3H — xanthen - 9-yl]phenyl}formamido)ethyl]sulfanyl} — 2 - cyanoeth — 1 - en yl]phenyl}methyl) - 3,3,6,6 - tetramethyl - 1 — thiacyclohept yn ium oroacetate (46): TFA' 8+ I I I I ACN, DIPEA 46a: 2-(6-(Dimethylamin0)(dimethyliminio)-3H-Xanthenyl)((2-mercaptoeth bam0yl)benzoate.
To a solution of TAMRA-5’-COOH (1 eq., 68.3 mg, 0.159 mmol) in DMF (0.228 mL), HATU (1 eq., 60.3 mg, 0.159 mmol), DIPEA (6 eq., 123 mg, 0.157 mL, 0.952 mmol) and cystamine dichloride (5 eq., 178 mg, 0.793 mmol) were subsequently added; the obtained solution mass was stirred overnight. A solution of DTT (5 eq., 122 mg, 0.118 mL, 0.793 mmol) in DCM (0.911 mL) was added to the reaction mass, the stirring continued for 2 hours. Solvents were evaporated; the ed crude mass was purified by HPLC to yield 463 (33.5 mg, 0.0555 mmol, 35 %) as a dark-Violet solid. 1H NMR (400 MHz, DMSO-ds) 89.06 (t, J = 5.4 Hz, 1H, 8.70 (d, J = 1.8 Hz, 1H), 8.30 (dd, J = 1.8, 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.08-7.02 (m, 4H), 6.95 (s, 2H), 3.52-3.42 (m, 2H), 3.26 (s, 12H), 2.72 (dt, J = 6.8, 8.0 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) 8166.0, 164.7, 156.8, 156.6, 135.9, 131.2, 130.6, 114.6, 96.3, 42.9, 40.5, 23.3; HR—ESI-MS: C27H27N3O4S: 489.1722; found 489.1723. 46: 1-({4-[(1Z){[2-({3-Carboxylato-4—[6-(dimethylamino)-3— (dimethyliminiumyl)-3H-xanthenyl]phenyl}formamido)ethyl]sulfanyl} cyanoeth-l-enyl]phenyl}methyl)-3,3,6,6—tetramethylthiacycloheptynium trifluoroacetate (TAMRA-APN-TMTI).
WO 2015001117 66 A solution of 45 (1 eq., 6.74 mg, 0.016 mmol) in ACN (1 mL) was mixed with a solution of 46a (1 eq., 9.64 mg, 0.016 mmol) in DMF (1 mL). DIPEA (5 eq., 132 uL, 0.08 mmol) was then added and ed reaction mass was injected into HPLC after 5 minutes of reaction to yield 46 (11.9 mg, 0.0149 mmol, 93 %) as a dark-violet solid. 1H NMR (400 MHz, DMSO—ds) 88.98 (t, J = 5.40 Hz, 1H), 8.30 (d, J = 8.28 Hz, 1H), 8.10 - 8.20 (m, 1H), 7.85 (s, 1H), 7.57 - 7.69 (m, 4H), 6.97 - 7.15 (m, 5H), 6.07 (s, 1H), 4.85 (s, 2H), 2.81 - 2.90 (m, 4H), 3.28 (br. s., 16H), 1.25 (s, 6H), 1.05 (s, 6H); 13C NMR (101 MHz, DMSO-d6) — not informative (low resolved signals); HR-ESI-MS: C47H49N4O4S2+, 797.31897; found 797.32739. 1 - ({4 — [2 - Cyano — 1 - [(2 — {4 - [(E) - 2 — [4-(dimethylamino)phenyl]diazen — 1 - yl]benzenesulfonamido}ethyl)sulfanyl]eth en-l - yl]phenyl}methyl) - 3,3,6,6 - tetramethyl thiacyclohept — 4 - yn — 1 - ium (47, BHQZ-APN- TMTI): TfO \\ 3+ <j // TFA N SH N” HN—g N O \‘o/N N l | \ 47a 0=S=O N\\ / rim INI ACN, DIPEA s/\/ 47a: (E)((4-(Dimethylamino)phenyl)diazenyl)-N- (2-mercaptoethyl)benzenesulfonamide.
To a cooled to 0°C solution of Dabsyl chloride (1 eq., 100 mg, 0.309 mmol) in dry ACN (3 mL), TEA (7 eq., 218 mg, 0.3 mL, 2.16 mmol) and cystamine ochloride (5 eq., 347 mg, 1.54 mmol) were subsequently added. After 2 hours of ng, DTT (6 eq., 285 mg, 0.275 mL, 1.85 mmol) was added to the reaction mass. The ed solution was stirred for another 2 hours, evaporated and the obtained crude product was purified by flash chromatography (cyclohexane-EtOAc) to yield 473 (105.9 mg, 94%) as an orange solid.
W0 20151001117 67 47: 1-({4-[(2-Cyano[(2-{4-[(E)[4- hylamino)phenyl]diazen yl]benzenesulfonamido}ethyl)sulfanyl]eth-l-en yl]phenyl}methyl)-3,3,6,6-tetramethyl thiacyclohept—4—ynium trifluoroacetate.
The same procedure as for the sis of the 46. Yield: 94%. 1H NMR (400 MHz, DMSO—ds) 58.03 (t, J = 4.89 Hz, 1H), 7.91 (d, J = 8.53 Hz, 2H), 7.80 - 7.87 (m, J: 9.04 Hz, 2H), 7.72 - 7.79 (m, J: 8.53 Hz, 2H), 7.68 (s, 4H), 6.87 (d, J = 9.04 Hz, 2H), 6.08 (s, 1H), 4.86 (s, 2H), 3.92 (d, J = 12.05 Hz, 2H), 3.84 (d, J = 12.30 Hz, 2H), 3.10 (s, 6H), 2.71 - 2.87 (m, 4H), 1.32 (s, 6H), 1.17 (s, 6H); 13C NMR (101 MHz, DMSO-ds) 5160.2, 158.6, 158.3, 155.1, 153.7, 143.1, 140.3, 136.8, 131.9, 131.3, 129.5, 128.2, 125.9, 122.8, 117.2, 112.1, 106.4, 99.4, 60.0, 43.3, 42.9, 34.5, 26.4, .3; HR—ESI-MS: C36H42N50283+, 672.24951; found 672.25042.
Compounds 46-47 can be used for the preparation of compounds otherwise not accessible (TMTI).
WO 2015001117 68 2014/064387 -(3—{4—[1-(4 - {[4 - (2 - Cyanoeth-l - yn nyl]carbamoyl}butyl) - 1H - 1,2,3 — triazol - 4-yl]butanamido}propyl) — 2 - [(E) - 2 — [4 - hydroxy — 2 - (2 — {2 - [2 - (2 - {5 - [(4S) 0X0 - hexahydro - 1H - thieno[3,4 - d]imidazolidin - 4-yl]pentanamido}ethoxy)ethoxy]ethoxy}ethoxy)phenyl]diazen yl]benzoic (48, APN-HAZA-biotin): OH 01(1ng/,NN goA/OI o 44, CuSO4-5H20 sodium ascorbate O —’ 1 NH DMSOIHZO NH O \\ O \\ / IIN “I“ s | | ML? ‘71/NH HN O 0 48a 48 Compound 48 can be used for purification and/or immobilization according to the invention. 4821: 2-(6-(Dimethylamino)(dimethyliminio)-3H-Xanthenyl)((2-mercaptoeth yl)carbamoyl)benzoate.
This compound was synthesised following the usly reported protocol. 48: 5-(3—{4—[1-(4—{[4-(2-Cyanoethynyl)phenyl]carbamoyl}butyl)-1H—1,2,3— triazolyl]butanamido}propyl)[(E)[4-hydroxy(2-{2-[2-(2-{5-[(4S)oxo- hexahydro-1H-thieno[3,4-d]imidazolidin WO 2015001117 69 yl]pentanamido}ethoxy)eth0xy]ethoxy}ethoxy)phenyl]diazenyl]benzoic (APN- HAZA-biotin).
To a solution of 483 (1 eq., 10 mg, 0.0123 mmol) and 77 (1 eq., 3.12 mg, 0.0123 mmol) in DMSO (0.472 mL), solution of sodium ascorbate (10 eq., 24.4 mg, 0.123 mmol) and 5H20 (5 eq., 15.4 mg, 0.0617 mmol) in water was added. The obtained reaction mass was degassed and stirred ght at 25 °C. The reaction mass was directly purified by HPLC to give 48 (8.3 mg, 0.0078 mmol, 63 %) as a yellow solid. 1H NMR (400 MHz, METHANOL—d4) 57.78 (br. s., 2H), 7.72 (d, J = 8.28 Hz, 1H), 7.67 (s, 1H), 7.58 (d, J = 8.78 Hz, 2H), 7.50 (d, J = 8.78 Hz, 2H), 7.29 (d, J = 8.28 Hz, 1H), 7.25 (d, J: 8.53 Hz, 1H), 6.29 (d, J: 7.78 Hz, 1H), 3.70 - 3.81 (m, 8H), 3.62 (d, J = 4.77 Hz, 2H), 3.58 (d, J: 5.02 Hz, 2H), 3.48 - 3.53 (m, 2H), 3.41 - 3.48 (m, 2H), 3.01 - 3.07 (m, 6H), 2.89 - 3.00 (m, 10H), 2.78 (dd, J: 4.89, 12.93 Hz, 1H), 2.52 - 2.67 (m, 8H), 2.33 (t, J: 7.28 Hz, 2H), 2.09 - 2.19 (m, 2H), 1.81 - 1.91 (m, 4H), 1.65 - 1.78 (m, 2H), 1.52 - 1.63 (m, 2H), 1.42 - 1.52 (m, 1H), 1.23 - 1.31 (m, 1H); HR-ESI—MS: C56H67N110iiS, 7422; found 1077.45931. 2-((4-(cyanoethynyl)phenyl)amino)-N,N,N-trimethyloxoethan-l-aminium trifluoroacetate (49): o N+\ >_/ / O \N‘ CI' \ N— — NH2 —> N: : NH DIPEA, DMF TFA. 7 49 49a: 2-chl0r0-N,N,N-trimethyl-Z-oxoethan-I-aminium.
Synthesised as previously described by Vassel and Skelly (10.1002/0471264180.os035.09). 49: 2-((4-(cyanoethynyl)phenyl)amino)-N,N,N-trimethyloxoethan-l-aminium trifluoroacetate.
To a solution of 3-(4-aminopheny1)propynenitrile (1 eq., 66.3 mg, 0.466 mmol) and DIPEA (1.1 eq., 66.3 mg, 0.0848 mL, 0.513 mmol) in DMF (1 mL), a cooled to -20 “C solution of (2-chlorooxoethyl)trimethy1azanium chloride (1.1 eq., 88.3 mg, 0.513 mmol) in DMF (1 mL) was added. The obtained reaction mass was stirred at 25 °C for W0 20151001117 70 2014/064387 hours, purified by RP-flash chromatography to give 49 as a yellowish solid (39 mg, 0.110 mmol, 24%). 1H NMR (400 MHz, ACETONITRILE-ds) 811.14 (br. s., 1H), 7.70 = 8.78 - 7.83 (m, J Hz, 2H), 7.56 = 8.78 Hz, 2H), 4.33 - 7.70 (m, J (s, 2H), 3.28 (s, 9H); 13C NMR (101 MHz, ACETONITRILE-ds) 8163.5, 142.5, 135.8, 121.2, 113.5, 106.5, 84.4, 66.3, 63.1, 55.2; ESI-MS: N30+ [M]+, 242.13; found 242.13. 3-(1-(1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazolyl)-2,5,8,11,14- pentaoxaheptadecanamid0)pentanedioic acid (50): _ _ N: \ N_ _ N —> \ \ o/\/0\/\ /\/0\/\o o N 0 OH A solution of 43 (1 eq., 5.65 mg, 0.0336 mmol) in DMSO (0.0331 mL), a solution of di- acid-alkyne (1 eq., 14.6 mg, 0.0336 mmol) in water (0.0331 mL) was added. A on of copper sulfate pentahydrate (0.1 eq., 0.839 mg, 0.00336 mmol) in minimum ammount of water was added to the obtained reaction mass followed by the addition of a solution of sodium ascorbate (0.5 eq., 3.33 mg, 0.0168 mmol) in minimum ammount of water. The addition repeated after 30 minutes until complete dissapearance of the strasting material (2 times overall). Excess of water was evaporated to vacuo (no heating should be used, otherwise hydrolysis product starts to appear), the ed crude mass was purified by HPLC after the filtration of copper salts through a seringe filter to give 50 (11 mg, 8 mmol, 54%) as a white solid. 1H NMR (400 MHz, ITRILE-dg) 88.41 (s, 1H), 7.95 - 8.02 (m, 2H), 7.88 - 7.94 (m, 2H), 6.86 (d, J = 7.78 Hz, 1H), 4.71 (s, 2H), 4.49 (td, J = 6.71, 8.41 Hz, 1H), 3.68 = 2.42, 4.96 - 3.76 (m, 3H), 3.49 - 3.68 (m, 21H), 2.53 - 2.64 (m, 5H), 1.97 (td, J Hz, 15H); 13C NMR (101 MHz, ACETONITRILE-ds) 8188.7, 173.3, 161.6, 146.5, 135.1, 131.3, 130.8, 122.5, 121.0, 117.9, 115.4, 64.2, 40.4, 39.5, 36.7, 30.6; ESI-MS: C28H34N5010‘ [M-H]‘, 600.21; found 600.23.
W0 20151001117 71 N1,NS-bis(23-amino-3,6,9,12,15,18,21-heptaoxatricosyl)(1-(1-(4- (cyanoethynyl)phenyl)-1H-1,2,3-triazolyl)-2,5,8,11,14- pentaoxaheptadecanamido)pentanediamide (51): NEG—N3 —> 0%HN ”NOWO/VOV\O/\/o\/\O/\/o\/\NH2 jgO 43 51 A solution of 43 (1 eq., 1.64 mg, 0.00973 mmol) and PEG-Alkyne (1 eq., 11 mg, 0.00973 mmol) in DMSO was added to a mixture of DMSO (0.00958 mL) and water (0.00958 mL). A solution of copper sulfate pentahydrate (0.1 eq., 0.243 mg, 0.000973 mmol) in m ammount of water was added to the obtained reaction mass ed by the addition of a solution of sodium ascorbate (0.5 eq., 0.964 mg, 0.00487 W0 20151001117 72 mmol) in minimum t of water. The reaction mass was d and purified by HPLC to give 51 (5 mg, 0.003839 mmol, 39%) as a colorless liquid.
ESI-HRMS: C60H103N9022, 1301.72177; found 1301.72204.
N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)(1-(4-(cyanoethynyl)phenyl)- ,3-triazolyl)-2,5,8,11,14-pentaoxaheptadecanamide (52): H2N\L ”N o N\N\ N;QN3 N —’ NWWOV\ONOV\O/\/O\j A solution of zidophenyl)propynenitrile (1 eq., 14.9 mg, 0.0886 mmol) in DMSO (0.0872 mL), a solution of PEG-alkyne (1 eq., 50.2 mg, 0.0886 mmol) in water (0.0872 mL). To the ed mixture a solution of copper sulfate pentahydrate (0.1 eq., 2.21 mg, 0.00886 mmol) in minimum ammount of water was added followed by the addition of a solution of sodium ascorbate (0.5 eq., 8.77 mg, 0.0443 mmol) in minimum ammount of water. The addition of copper sulfate pentahydrate (0.1 eq., 2.21 mg, 0.00886 mmol) and sodium ascorbate (0.5 eq., 8.77 mg, 0.0443 mmol) were repeated after 30 minutes if starting azide was still present. Excess of water was evaported to vacuo, the crude product was purified by HPLC to give 52 (55 mg, 0.07485 mmol, 84%) as a colorless oil. 1H NMR (400 MHz, ACETONITRILE-ds) 88.42 (s, 1H), 7.95 = 9.03 Hz, - 8.02 (m, J 2H), 7.86 - 7.95 (m, J: 8.78 Hz, 2H), 7.30 (br. s., 2H), 7.19 (br. s., 1H), 4.71 (s, 2H), 3.73 - 3.79 (m, 2H), 3.65 - 3.73 (m, 7H), 3.54 - 3.65 (m, 29H), 3.51 (t, J: 5.40 Hz, 2H), 3.34 (q, J= 5.35 Hz, 2H), 3.13 (d, J: 4.52 Hz, 2H), 2.37 - 2.46 (m, 2H), 1.92 - 2.01 (m, 5H); 13C NMR (126 MHz, CHLOROFORM-d) 8177.5, 151.3, 144.6, 140.7, 140.7, W0 20151001117 73 140.6, 127.2, 126.0, 125.9, 125.8, 122.4, 110.5, 87.2, 75.4, 75.3, 75.3, 75.2, 75.2, 75.1, 75.1, 75.1, 75.0, 74.9, 74.8, 74.8, 72.2, 71.9, 68.9, 68.5, 45.0, 44.2, 41.6; ESI-MS: C35H55N6011+ [M+H]+, 735.39; found 735.20. 1-(4-(cyan0ethynyl)phenyl)(3-(dimethylamino)propyl)urea (53): CI 0 CI C'>k JL x0 CI 0 0 CI //N I TEA / HN N —’ 2 : :N W + W \ THF,r.t.,2hours \NMNJLN I H H 7 53 To a solution of triphosgene (1 eq., 56.3 mg, 31.6 uL, 0.19 mmol) in THF (0.404 uL) was added a solution of 3-(4- aminopheny1)propynenitrile (3 eq., 80.9 mg, 0.569 mmol) in THF (0.404 uL). Then triethylamine (6 eq., 115 mg, 158 uL, 1.14 mmol). The mixture was stirred for 5 min and then 3-dimethylaminopropylamine (3 eq., 58.1 mg, 71.8 uL, 0.569 mmol) and triethylamine (2 eq., 38.4 mg, 52.7 uL, 0.379 mmol) was added in THF (0.404 uL). The reaction mixture was d for 10 minutes and then concentrated. The obtained residue was purified by HPLC to give 53 (47 mg, 0.1764 mmol, 93%) as a white solid. 1H NMR (400 MHz, METHANOL-d4) 87.45 - 7.50 (m, 2H), 7.41 - 7.45 (m, 2H), 3.23 - 3.25 (m, 1H), 3.04 - 3.11 (m, 2H), 2.80 (s, 6H), 1.77 - 1.91 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8156.5, 143.7, 134.4, 118.0, 109.3, 105.0, 83.7, 61.0, 55.2, 42.1, 35.9, 25.3; ESI-MS: N4O+ [M+H]+, 271.16; found 271.15. 3-((3-(3-(4-(cyanoethynyl)phenyl)ureid0)propyl)dimethylammonio)propane ate (54): N\\ N\\ \\ 1,3-propanesultone, \\ o Ncho3 o NJLNMN/—> o IPA, 80 00. 3 hours N N/\/\N+’\/\s’/ H H | _ 53 54 53 (1 eq., 27 mg, 0.0702 mmol) and 1,3-propanesultone (1.1 eq., 9.44 mg, 8 mL, 0.0773 mmol) were dissolved in IPA (0.5 mL) and refluxed for 3 h. The reaction mixture was cooled to room temperature and the precipitate was filtered and washed WO 01117 74 with cold distilled water to remove any unreacted propanesultone to give 54 (27 mg, 0.0688 mmol, 98 %) as a white solid. 1H NMR (400 MHz, DEUTERIUM OXIDE) 57.45 - 7.67 (m, 2H), 7.33 (br. s., 2H), 3.61 (br. s., 4H), 3.53 (br. s., 1H), 3.39 (br. 5., 2H), 3.31 (br. s., 2H), 3.25 (br. s., 2H), 3.03 (br. s., 7H), 2.89 (br. s., 7H), 2.13 (br. s., 1H), 1.89 (br. s., 7H), 1.08 (br. s., 2H); ESI-HRMS: C18H24N404S, 392.15183; found 254.Compounds 49-54 can be used for a conjugation method according to the invention, for instance for changing ADME parameters (solubilizing agents). 4-(hydroxymethyl)-1H-1,2,3-triazolyl)phenyl)propiolonitrile (55): sodium ascorbate,Cu| HO/\|%\ _ _ N3 _— :N N _ _N N: / water, THF, 25 “C, 2 hours N 43 55 3-(4-azidophenyl)propynenitrile (1 eq., 300 mg, 1.78 mmol) , 2-propynol (2 eq., 200 mg, 0.211 mL, 3.57 mmol) were solubilized in THF (9 mL). To this mixture was added a solution of copper sulfate ydrate (10 %, 44.5 mg, 0.178 mmol) in 1.5 mL of water followed by the solution of sodium ascorbate (0.5 eq., 176 mg, 0.892 mmol) in 1.5 mL of water. The resulting solution was stirred for 2h and then concentrated on rotary evaporator. The residue was extracted with DCM. The organic layer was washed with NH4C1 (sat) and water, dried over MgSO4 and then evaporated. The residue was resolubilised in DCM and the product was filtered to give 55 (23.28 mg, 0.08108 mmol, 92%) as a slightly yellowish solid. 1H NMR (400 MHz, MeOD) 8 8.58 (s, 1H), 8.04 (d, J = 7.4 Hz, 2H), 7.93 (d, J = 7.4 Hz, 2H), 4.77 (s, 2H); ESI-MS: C12H9N4O+ [M+H]+, 225.08; found 225.05. 3-(4-(4-(bromomethyl)-1H-1,2,3-triazolyl)phenyl)propiolonitrile (56): HOyr: PBr3 : :N Br/Y\N : :N NQN’ THF, 25 00, 15 hours NQN 55 56 55 (1 eq., 19.8 mg, 0.0881 mmol) was dissolved in THF (1 mL) under nitrogen at room temperature and 3-{4-[4-(bromomethyl)—1H-1,2,3-triazolyl]phenyl}prop ynenitrile (23.3 mg, 0.0811 mmol, 92 %) was added. The reaction mixture was stirred at room WO 2015001117 75 temperature for 15 hours. Solvents were evaporated, the crude product was d by HPLC to give 56 (23.3 mg, 0.0811 mmol, 92 %) as a white solid. 1H NMR (400 MHz, ITRILE-ds) 88.44 (s, 1H), 7.78 - 8.03 (m, 5H), 4.74 (s, 2H); 13C NMR (101 MHz, CHLOROFORM-d) 8150.9, 144.3, 140.6, 140.5, 126.0, 125.9, 110.4, 87.1, 68.5, 27.0; ESI-MS: C12H8BrN4+ [M+H]+, 286.99; found 287.08.
Compound 56 can be used for a bioconjugation method according to the invention. 1-({1-[4-(cyanoethynyl)phenyl]-1H-1,2,3-triazolyl}methyl)-3,3,6,6-tetramethyl- 4,5-didehydro-2,3,6,7-tetrahydrothiepinium oroacetate (57): F 3 Br/\F\ TMTH, LIOTf. 3+ N _— :N \\ N — _N NQN’ DCM, water, 25 "C, 150 hours 56 57 To a degassed solution of 4-(bromomethyl)-1H—1,2,3-triazol-l-yl]phenyl}prop ynenitrile (1 eq., 19.6 mg, 0.0684 mmol) and TMTH (1.89 eq., 21.7 mg, 0.129 mmol) in DCM (0.982 mL), a solution of lithium e (10 eq., 106 mg, 0.684 mmol) in water (0.982 mL) was added. The obtained biphasic mixture was vigorously mixed for 2 days at 25 ”C. Phases were separated, organic phase was washed with DCM (5x2 mL).
United organic fractions were evaporated and the crude was purified by HPLC to give 57 (23.7 mg, 0.0451, 66%) as a white solid.
ESI-HRMS: C22H23N4S, 375.16434; found 375. 16497.
Compound 57 can be used for click-chemistry (strain promoted click) according to the invention. 4-(((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4—yl)methyl)carbamoyl)(6- (dimethylamino)-3—(dimethyliminio)-3H-xanthenyl)benzoate (58): WO 20151001117 76 N‘ ‘ AFN-NB, sodium ascorbate, Cul 0, 25 °C, 30 mins 2-[6-(dimethylamino)—3-(dimethyliminiumyl)—3H-xantheny1]-4—[(propyn yl)carbamoyl]benzoate (1 eq., 52.8 mg, 0.113 mmol) and 3-(4-azidophenyl)prop rile (1 eq., 19 mg, 0.113 mmol) were solubilized in THF (1 mL). H20 (1 mL) was added to the obtained reaction mixture followed by the addition of solutions of Copper Sulphate pentahydrate (10 %, 2.82 mg, 0.0113 mmol) and sodium ascorbate (50 %, 11.2 mg, 0.0565 mmol) in minimum amount of water (separately). The obtained reaction mixture was stirred for r 30 minutes, evaporated and purified by HPLC to give 58 (66.8 mg, 0.105 mmol, 93 %) as a dark-violet solid. 1H NMR (400 MHz, METHANOL-d4) 88.51 (s, 1H), 8.33 (d, J = 8.28 Hz, 1H), 8.16 (dd, J = 1.51, 8.28 Hz, 1H), 7.86 — 8.02 (m, 2H), 7.64 - 7.86 (m, 3H), 7.02 - 7.12 (m, 2H), 6.84 - 7.02 (m, 4H), 4.58 - 4.71 (m, 2H); ESI-HRMS: C37H29N7O4, 635.22811; found 635.22861. 3-(9-(diethylamino)0X0-5H-benzo[a]phenoxazinyl)propiolonitrile (59): OH O\\ J<F S F O \O\ propargylic alcohol, TEA’ TfCI L11 9N O DIPEA, PdCI2(PPh3)2 N —> N O O L 2 hours K N O O OH N N O MnOz‘ MgSO4, NH, L \ —, N\ THF, 25 00, 15 mins N o 0 L Q: Q K NK 0 0 59a 59 WO 20151001117 77 59b: 8-(diethylamino)—3-hydroxy-12Hoxaazatetraphenone (1 eq., 65 mg, 0.194 mmol) was dissolved in dry DCM (2 mL) and cooled to 5 °C. Then TEA (1.2 eq., 23.6 mg, 0.0324 mL, 0.233 mmol) was added followed by the addition of TfCl (1.2 eq., 39.3 mg, 0.0249 mL, 0.233 mmol). The on of Tf20 was repeated untile complete disappearance of the starting material. The solvent was evaporated and the residue was treated with water. The precipitate was d off and washed with water and heptane to give the desired product (76 mg, 0.163 mmol, 84 %) as a dark-Violet solid.
ESI-HRMS: F3N205S, 466.08103; found 466.08221. 593: Under an inert atmosphere, DIPEA (2 eq., 16.1 mg, 0.0206 mL, 0.124 mmol) and Propargylic alcohol (1.5 eq., 5.23 mg, 0.00551 mL, 0.0933 mmol) were added to a on of 59b (1 eq., 29 mg, 0.0622 mmol), PdC12(PPh3)2 (5 %, 2.18 mg, 0.00311 mmol) and CuI (10 %, 1.18 mg, 0.00622 mmol) in DMF (1 mL). After stirring for 2 hours at 90 °C, the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (DCM-MeOH from 100-0 to 80- 20).
ESI-HRMS: C23H20N203, 372.14739; found 372.14735. 59: To the solution of 59a (1 eq., 10 mg, 0.0269 mmol) in THF (0.121 mL) was added MgSO4 (15 eq., 48.5 mg, 0.403 mmol), NH3 (4 eq., 2 M, 0.0537 mL, 0.107 mmol), and Mn02 (15 eq., 35 mg, 0.403 mmol).The reaction e was stirred at r.t. for 15 mins and followed by HPLC. After completion the mixture was filtered through Celite and washed thoroughly with THF. Evaporation of the e gave crude 59 (9.47 mg, 0.0258 mmol, 96 %) which was purified by HPLC. 1H NMR (400 MHz, CHLOROFORM—d) 88.91 (s, 1H), 8.26 (d, J = 8.03 Hz, 1H), 7.79 (dd, J: 1.51, 8.03 Hz, 1H), 7.62 (d, J: 9.29 Hz, 1H), 6.74 (dd, J: 2.63, 9.16 Hz, 1H), 6.51 (d, J = 2.51 Hz, 1H), 6.35 (s, 1H), 5.31 (s, 2H), 3.49 (q, J = 7.11 Hz, 4H), 1.09 - 1.36 (m, 6H); ESI-HRMS: C23H17N302, 367.13208; found 367.13145.
(E)(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)propiolonitrile (60): H N : :N —> z 2 ’ N: — N \ 7 60 7 (1 eq., 167 mg, 1.17 mmol) was dissolved in acetonitrile (2.58 mL). To this stirred e was added Isoamyl nitrite (1.5 eq., 206 mg, 0.237 mL, 1.76 mmol), stirring W0 20151001117 78 continued for another 2 minutes, then dimethylaniline (1.1 eq., 156 mg, 0.165 mL, 1.29 mmol) was added. The resulting reaction mixture was stirred overnight (turned red), evaporated and purified by flash chromatography (DCM, first peak) to give 60 (120 mg, 0.437 mmol, 37 %) as red solid. 1H NMR (400 MHz, FORM-d) 57.91 (d, J = 9.03 Hz, 2H), 7.82 = - 7.88 (m, J 8.53 Hz, 2H), 7.62 = 8.53 Hz, 2H), 6.77 (d, J = 9.03 Hz, 2H), 3.08 - 7.77 (m, J - 3.18 (m, 6H); 13C NMR (101 MHz, CHLOROFORM-d) 5154.7, 153.2, 143.7, 134.4, 125.8, 122.5, 117.3, 111.6, 105.6, 83.3, 64.2, 40.3; ESI-HRMS: C17H14N4, 274.12185; found 274.12247.
Compounds 58-60 can be used for a detection method method according to the invention. utyl ((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol—4-yl)methyl)carbamate (61) : N: : cBu‘I’Z‘ZEE‘I‘nrgaVS'EElS—ée THF waten 25°C 30 minsN\©‘N/\’/\NHBoc To a solution of 43 (1 eq., 51.5 mg, 0.306 mmol) and boc-propargylamine (1 eq., 47.5 mg, 0.306 mmol) in THF (2 mL) were added H20 (1 mL) and solution of CuSO4 (10 %, 4.89 mg, 0.0306 mmol) and sodium ate (50 %, 30.3 mg, 0.153 mmol) in water (50 uL each). Stirring continied for 10 minutes, one more portion of CuSO4 (10 %, 4.89 mg, 0.0306 mmol) and sodium ascorbate (50 %, 30.3 mg, 0.153 mmol) was added.
After another 15 minutes of stirring, EtzO (15 mL) and NH4C1 (sat, 10 mL) were added.
Organic phase was washed two more times with NH4C1 (sat, 10 mL), dried over MgSO4 and evaporated to give 61 (98 mg, 0.303 mmol, 99 %) as a yellow solid. Used without further ation. 1H NMR (400 MHz, CHLOROFORM-d) 67.97 (s, 1H), 7.74 = 8.78 Hz, - 7.81 (m, J 2H), 7.67 = 8.78 Hz, 2H), 4.41 - 7.74 (m, J (d, J = 6.02 Hz, 2H), 1.32 - 1.41 (m, 9H); 13C NMR (101 MHz, CHLOROFORM-d) 6139.0, 135.1, 120.4, 117.8, 107.2, 105.1, 81.3, 64.5, 28.4; : C17H18N502+ [M+H]+, 323.14; found 323.13.
W0 20151001117 79 (1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazolyl)methanaminium trifluoroacetate (62): —>*H3N/W/\N < >: 1‘":ka N\©~NMNHBOC DCM 25 °,c 15 hours To a solution of 61 (1 eq., 21.5 mg, 0.0665 mmol) in DCM (1 mL) was added TFA (20 eq., 151 mg, 0.0988 mL, 1.33 mmol). The obtained reaction mixture was left overnight at room temperature (or 2 hours at 37 °C) to the targeted product (22.4 mg, 0.0665 mmol, 100 %) after the evaporation of all volatile compounds. 1H NMR (400 MHz, METHANOL-d4) 88.74 (s, 1H), 7.99 - 8.15 (m, 2H), 7.85 — 7.97 (m, 2H), 4.25 - 4.45 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) , 138.9, 135.2, 122.3, 120.3, 117.7, 104.4, 81.1, 62.9, 34.0; ESI-MS: C12H10N5+ [M]+, 227.09; found 227.10.
N-((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazolyl)methyl)(3- (trifluoromethyl)-3H-diazirinyl)benzamide (63): ‘H3N o HATU DIPEA ”MN _, «N DMF, 25 °C. 10 mins ii NzN’ A solution of 4-[3-(trifluoromethyl)-3H-diazirinyl]benzoic acid (1 eq., 57.8 mg, 0.251 mmol), HATU (1 eq., 95.5 mg, 0.251 mmol), and DIPEA (3 eq., 97.4 mg, 0.125 mL, 0.753 mmol) in DMF (2 mL) was added onto 62 (1 eq., 84.7 mg, 0.251 mmol). The obtained reaction mass was d for 10 minutes and purified by HPLC to give the targeted compound (76.5 mg, 0.176 mmol, 70 %) as a white solid.
ESI-HRMS: C21H12F3N7O, 435.10554; found 435.10512.
Compound 63 can be used for a labeling method according to the invention, such as for abeling of proteins. wo 2015;001117 80 tert-Butyl (2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethyl)carbamate (64) : F: K k TEA H N s —> 2 \/\s/ \ANAO ACN,25°C, 10mins CI 0 To a solution of 23 (1 eq., 36 mg, 0.143 mmol) in acn (1 mL) was added a solution of utyl N—{2-[(2-aminoethyl)disulfanyl]ethyl}carbamate (1 eq., 36 mg, 0.143 mmol) and DIPEA (2.12 eq., 39.1 mg, 0.05 mL, 0.303 mmol) in ACN (1 mL). The obtained reaction mixture was left for 10 minutes, then solvents were evaporated and the crude t was purified by flash chromatography to give the targeted product (29.3 mg, 0.0723 mmol, 51 %) as a yellowish solid. 1H NMR (400 MHz, METHANOL-d4) 57.74 = 8.53 Hz, 2H), 7.58 - 7.84 (m, J - 7.74 (m, J: 8.53 Hz, 2H), 3.60 (t, J = 6.78 Hz, 2H), 3.23 - 3.35 (m, 2H), 2.85 (t, J = 6.78 Hz, 2H), 2.71 (t, J: 6.90 Hz, 2H), 1.32 (s, 9H); 13C NMR (101 MHz, METHANOL-d4) 8167.3,157.0,137.3, 133.5, 120.1, 104.4, 81.5, 78.8, 63.1, 39.3, 39.1, 37.8, 27.4, 26.6; ESI-MS: C19H24N303S2+ [M+H]+, 406.12; found 406.10. 2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethan-l-aminium trifluoroacetate (65): WO 20151001117 81 HN o TFA F38k —. F DCM, ACN, 0 NH °c, 24 hours B || 3 INI W e4 65 To a solution of 64 (1 eq., 29.3 mg, 0.0723 mmol) in ACN—DCM mixture (1 mL of each solvent) was added TFA (10 eq., 82.4 mg, 0.0537 mL, 0.723 mmol). The obtained on mixture was stirred for 24 hours, and evaporated to give the targeted compound 65 (30 mg, 0.0715 mmol, 99 %) as a colorless liquid.
ESI-HRMS: C14H16N3OSZ+, 306.07293; found 306.07312. 2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethan-l-aminium trifluoroacetate (66): *H3N HO ,0 [co with own, 5,: ~ umPM ,3” || ‘YO || \\N 62 66 62 (0.567 eq., 10.1 mg, 0.0299 mmol) solution in MeOH (0.0881 mL) was slowly added to a solution of 2-[4,7,10-tris(carboxymethyl)-1,4,7,l0-tetraazacyclododecanyl]acetic acid (1 eq., 21.4 mg, 0.0528 mmol) in water (0.661 mL). The mixture was cooled by ice and pH was adjusted to 5 using DIPEA (25.2 eq., 172 mg, 0.22 mL, 1.33 mmol). An aqueous solution of EDC (0.65 eq., 6.59 mg, 0.0344 mmol) was added se and d for 20 min with ice cooling. pH was raised to 8 using DIPEA and reacted for 30 min at room temperature. The end point of the reaction was monitored using HPLC.
WO 20151001117 82 ESI-HRMS: N907+, 609.26594; found 609.26417.
Compound 66 can be used as a chelating agent.
WO 2015001117 83 -((4-((4-(2-Cyano((2-(4-((4-((E)-(2,5-dimeth0xy((E)-(4-nitr0phe- nyl)diazenyl)phenyl)diazenylphenyl)- (methyl)amino)butanamid0)ethyl)thi0)vinyl)phenyl)amin0) oxobutyl)carbam0yl)(6-(dimethylamino)(dimethyliminio)-3H-Xanthen yl)benzoate (A): N\‘N o A I cystamine dichloride I HC| AcONa O O N\\N | —’ N2+CI Hzoacetone (11) N\\N TEA, HBTU, DMF/DOM Fast BlackK © OH BHQ-Z-SH NH2 —> HBTU, TEA, DMF DIPEA, ACN WO 2015001117 84 4-((4-((E)-(2,S-Dimethoxy((E)-(4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)( methyl)amino)-butanoic acid (BHQ-2).
Fast Black K hemi (zinc chloride) salt (practical grade, z30% dye content) (7.76 g) was suspended in cold water (150.0 mL, 0°C) and stirred for 20 minutes. The suspension was filtered, and the red on was added se to a cold (0 oC) mixture of 4- (methyl(pheny1)amino)butanoic acid (1.33 g, 6.88 mmol), concentrated hydrochloric acid (3.1 mL) and sodium acetate (3.6 g, 43.90 mmol) in acetone mixture (1:1) (150.0 mL). The reaction mixture was stirred at 10 °C for 15 minutes and at room temperature for 2 hours. Then the on crude was extracted with ethyl acetate (3x150 mL) and the combined organic layers were dried over Na2SO4. The crude t was purified by column chromatography on silica gel (100% EtOAc, then 100% DCM to DCM/MeOH (95:5)). BHQ-2 (1.36 g, 39%) was obtained as a dark Violet solid. 1H NMR (400 MHz, METHANOL-d4) 5 8.31 (d, J = 9.0 Hz, 2H), 8.00 (d, J = 9.0 Hz, 2H), 7.86 (d, J = 9.0 Hz, 2H), 7.45 (s, 1H), 7.40 (s, 1H), 6.77 (d, J = 9.0 Hz, 2H), 4.05 (s, 3H), 4.00 (s, 3H), 3.5 (t, J: 7.1 Hz, 2H), 2.36 (t, J: 7.1 Hz, 2H), 1.98-1.90 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 5 176.2, 157.1, 154.3, 153.0, 151.4, 149.0, 147.4, 145.0, 142.6, 126.9, 125.3, 124.2, 112.1, 101.7, 100.7, 57.2, 52.3, 39.0, 31.6, 22.9. 65b: 4-((4-((E)-(2,5-Dimethoxy((E)-(4-nitrophenyl)diazenyl)phenyl)diazenyl)phe nyl)(methyl)amino)-N-(2-mercaptoethyl)butanamide ((BHQ-2)-SH).
BHQ-2 (1 eq., 92.2 mg, 0.182 mmol) was dissolved in a mixture of DMF (5 mL) and DCM (10 mL). TEA (6 eq., 152 uL, 1.09 mmol) and cystamine dichloride (5 eq., 204 mg, 0.91 mmol) were added. The mixture was cooled to 0 °C and HBTU (1 eq., 69 mg, 0.182 mmol) was added. The solution was allowed to reach room temperature and d for 15 hours. When total conversion was reached, DTT (6 eq., 168 mg, 0.162 mL, 1.09 mmol) was added. After the resulting mixture has been stirred for 10 minutes at room temperature, the crude was diluted with saturated NaHCOs solution (75 mL) and extracted with EtOAc (2x50 mL). The organic layers were combined, washed with water (50 mL), brine (50 mL) and dried over Na2SO4. The crude product was purified by column chromatography on silica gel eOH from 100:0 to 95:5) to yield (BHQ-2)-SH (60.7 mg, 0.107 mmol, 59 %) as a dark Violet solid.
W0 20151001117 85 1H NMR (400 MHz, CHLOROFORM-d) 5 8.33 (d, J = 9.0 Hz, 2H), 8.0 (d, J = 9.1 Hz, 2H), 7.9 (d, J = 9.1 Hz, 2H), 7.42 (s, 1H), 7.42 (s, 1H), 6.75 (d, J = 9.00 Hz, 2H), 5.90 (t, J: 5.6 Hz, 1H), 4.06 (s, 3H), 4.01 (s, 3H), 3.49 (t, J: 7.4 Hz, 2H), 3.41 (dt, J: 6.2, 6.4 Hz, 2H), 2.64 (td, J = 6.4, 8.47 Hz, 2H), 2.24 (t, J: 7.4 Hz, 2H), 2.01-1.94 (m, 2H); 13C NMR (101 MHz, CHLOROFORM-d) 6 172.2, 156.6, 153.8, 152.4, 151.1, 148.5, 147.0, 144.7, 142.3, 126.4, 124.9, 123.7, 111.6, 101.2, 100.3, 57.0, 56.9, 51.8, 42.5, 38.7, 33.4, 24.9, 23.0; HR—ESI-MS: C27H31N705S, 07; found 565.2105. 67: 5-((4-((4-(Cyanoethynyl)phenyl)amino)oxobutyl)carbamoyl)(6-(dimethyla mino)(dimethyliminio)-3H-Xanthenyl)benzoate.
To a cooled to 0 °C degased solution of 62a (1 eq., 17.3 mg, 0.0507 mmol) and TAMRA-5’—COOH (1 eq., 21.8 mg, 0.0507 mmol) in DMF (1.4 mL), HBTU (1 eq., 19.2 mg) was added at 0 °C. Obtained reaction mass was stirred for 5 minutes and TEA was added. The reaction mass was stirred for 1 hour at 25 OC, evaporated and purified by HPLC to yield 65a (22 mg, 68%) as a dark—violet solid. 1H NMR (400 MHz, OL-d4) 8 8.8 (br. s, 1H), 8.7 (s, 1H), 8.08 = - 8.16 (d, J 8.2 Hz, 1H), 7.60 = 8.9 Hz, 2H), 7.49 = 8.9 Hz, 2H), 7.32—7.39 - 7.70 (d, J - 7.58 (d, J (d, J: 8.2, 1H), 8 7.01 (s, 4H), 6.93 (s, 2H), 3.48-3.58 (m, 2H), 3.26 (s, 12H), 2.44-2.54 (t, J = 7.17 Hz, 2H), .12 (m, 2H); 13C NMR (101 MHz, OL-d4) — not informative; HR—ESI-MS: 639.24817; found 639.24310.
A: 5-((4-((4-(2-Cyano-l-((2-(4-((4-((E)-(2,5-dimethoxy((E)-(4-nitrophenyl )diazenyl)phenyl)diazenylphenyl)- (methyl)amino)butanamido)ethyl)thio)vinyl)phenyl)amino) oxobutyl)carbamoyl)(6-(dimethylamino)(dimethyliminio)-3H-Xanthen yl)benzoate.
To a degased solution of BHQ-SH (1.13 eq., 2 mg, 0.00354 mmol) in DCM (0.5 mL), a degased solution of 67 (1 eq., 2 mg, 0.00313 mmol) in methanol (0.5 mL) was added.
TEA (4.6 eq., 2 uL, 0.0144 mmol) was added and the obtained on mass was left overnight at 25 oC. Solvents were evaporated; the crude product was solubilised in DMSO (0.5 mL) and purified by HPLC to give BHQ-APN—TAMRA (A, 2.7 mg, 0.00225 mmol, 72%) as dark—Violet solid. 1H NMR (400 MHz, METHANOL-d4) 8 8.63 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 8.8 Hz, 2H), 8.22 (dd, J = 7.8, 2.0 Hz, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 9.0 Hz, 2H), WO 2015001117 86 7.69 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 9.0 Hz, 2H), 6.75-6.86 (m, 8H), 5.48 (s, 1H), 4.02 (s, 3H), 3.92 (s, 3H), 3.58-3.63 (m, 2H), 3.45—3.52 (m, 2H), 3.21 (s, 12H), 3.16 (t, J = 6.9 Hz, 2H), 3.08 (s, 3H), 2.71 (t, J = 6.5 HZ, 2H), 2.55 (t, f = 6.2 HZ, 2H), 2.22 (t, J = 6.9 HZ, 2H), 2.08—2.16 (m, 2H), 1.88- 1.96 (m, 2H), 1.61 (br.s, 1H); 13C NMR (101 MHz, METHANOL-d4) — not informative; HR-ESI-MS: C65H65N1201OSJr , 120546618; found 120546748. -((3-(3-((2-(4-((4-((E)-(2,5-Dimethoxy((E)-(4-nitrophenyl)diazenyl)phenyl)diaze nyl)phenyl)(me- thyl)amino)butanamido)ethyl)thio)-2,5-dioxopyrrolidinyl)propyl)carbamoyl)( methylamino)—3—(dimethyliminio)-3H-Xanthenyl)benzoate (B): E G 1. A toluene A Br/\/\NHBOC O , O O O 2. TFA, DCM N N 5—TAMRA TFA- —. —. —> K2003, DMF HBTU, TEA, DMF O O +HBN BocHN B-4 B-3 B-2 (BHQ—2)—SH (65b), DCMzMeOH B-4: 3a,4,7,7a-Tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione.
This compound was synthesised according to the previously described procedure. 845 W0 20151001117 87 B-3: tert-Butyl (3-((3aR,7aS)-1,3-dioxo-33,4,7,7a-tetrahydro-1H- 4,7-epoxyisoindol-2(3H)-yl)propyl)carbamate.
To the solution of B-4 (1 eq., 1.76 g, 10.7 mmol) and tert—butyl N—(3- bromopropyl)carbamate (2 eq., 5.07 g, 21.3 mmol) in DMF (20 mL), K2COs (1.2 eq., 1.77 g, 12.8 mmol) was added. The obtained reaction mass was heated at 50 °C for 18 hours. The solution was left to cool down; a solid residue was filtered and washed with DMF. United organic fractions were evaporated, hexane (50 mL) was added to the obtained slurry mass. Obtained suspensions were stirred for another hour, d and washed with hexane to give B-3 (3.36 g, 10.4 mmol, 98 %) as white solid. 1H NMR (400 MHz, CHLOROFORM-d) 8 6.49 (s, 2H), 5.23 (s, 2H), 3.52 (t, J = 6.5 Hz, 2H), 2.96 — 3.09 (m, 2H), 2.82 (s, 2H), 1.66-1.75 (m, 2H), 1.41 (s, 9H); 13C NMR (101 MHz, CHLOROFORM—d) 8 176.5, 155.9, 136.5, 81.0, 79.3, 47.5, 37.1, 36.0, 28.4, 27.8.
B-2: 1-(3-Aminopropyl)-1H-pyrrole-2,5-dione (TFA salt).
A solution of 66c (1 eq., 243 mg, 0.754 mmol) in toluene (25 mL) was refluxed for 3 hours. Toluene was evaporated; the obtained white crude product was resolubilised in DCM (5 mL), TFA (0.5 mL) was added. Stirring was continued for 2 hours until complete disappearance of a starting material (controlled by TLC). Solvent were ated after the reaction was quenched by ol (3 mL). Obtained l-(3- aminopropyl)—1H-pyrrole-2,5-dione (B-2, TFA salt, 190 mg, 94%) was used without fiarther purification. 1H NMR (400 MHz, METHANOL-d4) 5 6.76 (s, 2H), 3.52 (t, J = 6.7 Hz, 2H), 2.80- 2.88 (m, 2H), 1.76-1.88 (m, 2H); 13C NMR (101 MHz, METHANOL-d4) 8 170.6, 135.6, 38.5, 35.4, 28.0.
B-l: 2-(6-(Dimethylamino)(dimethyliminio)-3H-xanthenyl)((3-(2,5-dioxo-2, S-dihydro-IH-pyrrolyl)propyl)carbamoyl)benzoate -maleimide): To a solution of TAMRA-5’-COOH (1 eq., 71.5 mg, 0.166 mmol) in DMF (3.21 mL), TEA (2.5 eq., 57.7 uL, 0.415 mmol) and HATU (1.12 eq., 70.7 mg, 0.186 mmol) here added. Obtained reaction mass was stirred for another 5 minutes and B-2 (1 eq., 71.5 mg, 0.166 mmol) was added. ng continued for 25 s and the reaction mass was evaporated under reduced pressure to the volume of about 1 mL, and the reaction WO 2015001117 88 mass was purified by preparative HPLC to give Maleimide (B-l, 34.8 mg, 0.0615 mmol, 37 %) as a pink solid. 1H NMR (400 MHz, METHANOL-d4) 5 8.66 (d, J: 1.8 Hz, 1H), 8.14 (dd, J: 1.8, 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 9.5 Hz, 1H), 6.92 (dd, J = 9.5, 2.2 Hz, 2H), 6.81 (d, J = 2.2 Hz, 2H), 6.72 (s, 2H), 3.52 (t, J: 6.8 Hz, 2H), 3.34 (t, J: 7.0 Hz, 2H), 3.17 (s, 12H), .90 (m, 2H); 13C NMR (101 MHz, OL-d4) 8 172.5, 168.2, 167.4, 160.6, 159.0, 158.9, 138.1, 137.7, 137.6, 135.5, 132.9, 132.3, 132.0, 131.4, 115.6, 114.8, 97.5, 82.4, 41.0, 38.6, 36.4, 29.3. HR-ESI-MS: C32H30N406, 566.21653; found 566.21654.
B: 5-((3-(3-((2-(4-((4-((E)-(2,5-Dimethoxy((E)-(4-nitro- phenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)bu- tanamido)ethyl)thio)-2,5-dioxopyrrolidinyl)propyl)car— bamoyl)(6-(dimethylamino)(dimethyliminio)-3H-Xanthenyl)benzoate.
To a degased solution of BHQ—SH (1.15 eq., 4.6 mg, 0.00812 mmol) in DCM (0.5 mL), a degased solution of TAMRA-Maleimide (B-1) (1 eq., 4 mg, 3 mmol) in methanol (0.5 mL) was added. TEA (5 eq., 5 uL, 0.0353 mmol) was added and the obtained reaction mass was left overnight at 25 oC. Solvents were evaporated; the crude product was solubilized in DMSO (0.5 mL) and purified by HPLC to give B (7 mg, 0.00621 mmol, 88%) as dark-violet solid. 1H NMR (400 MHz, DMSO-d6) 8 8.91 (t, J: 6.1 Hz, 1H), 8.68 (s, 1H), 8.43 (d, J: 9.1 Hz, 2H), 8.31 (d, J: 8.9 Hz, 1H), 8.01-8.10 (m, 3H), 7.77 (d, J: 9.1 Hz, 2H), 7.60 (d, J = 7.8 Hz, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 6.99 (s, 3H), 6.89 (s, 1H), 6.85 (d, J: 9.1 Hz, 2H), 4.04 (dd, J = 3.9, 8.9 Hz, 1H), 3.98 (s, 3H), 3.92 (s, 3H), 3.41—3.45 (m, 2H), 3.27- 3.38 (m, 6H), 3.23 (m, 12H), 3.06 (s, 3H), 2.85-2.95 (m, 1H), 2.72-2.81 (m, 1H), 2.52- 2.56 (m, 2H), 2.17 (t, J= 7.3 Hz, 2H), 1.74-1.88 (m, 4H). HR-ESI-MS: C59H62N11O11S+ [M+H]+, 3455; found 1132.43384.
Example 2: Labeling of a cysteine derivative with compounds of the invention WO 20151001117 89 :N + —.
ACHN R|:/\)LNHBnNHAc R4 NHBn 1-12 AcCysNHBn General procedure To a vial containing 985 11L of PBS (1x, pH 7.6), were subsequently added 5 11L of the stock solution of benzamide (10 mM in water), 5 nL of the stock solution of arylpropiolonitrile (1-12, 10 mM in DMSO) and 5 ML of stock solution of AcCysNHBn (7m, 10 mM in DMSO). Aliquots of the reaction mixture (50 1.1L) were analyzed by HPLC (injection at 0 and 30 minutes of on). Areas under the peaks of the starting materials and hydrolysis products were normalized according to the area of the peak of the internal standard. s Obtained results are summed up in table 2 below, which presents the conversion of the compounds 1-12 in 30 minutes in presence of 7m at 50 11M concentration of each reagent and 25°C. The on is extremely sensitive to steric hindrances induced by substituents in ortho-position to propiolonitrile group (entries 1, 5, 8-9, 4) as well as to electronic effect of the substituent: -I and -M substituents increase (entries 10 and 12), while +M substituents decrease the reactivity of the compound (entries 3 and 7). .1% 70.4% * 46.8% Me 85.5% -I_———E- * ucts ** sion in 60 minutes. were observed; Table 2 Exam le 3: H drol ‘c stabilit of a com ound of formula I and com arison with phenylmaleimide WO 20151001117 90 To a vial containing 980 uL of PBS (1X, pH 7.6), were subsequently added 10 uL of a stock solution of benzamide and 10 uL of a stock solution of electrophile ACN : :N lmaleimide 1 or 11) to give final concentration of 1 mM (both internal standard and electrophile). ts of the reaction mixture (50 uL) were analyzed by HPLC for 5 hours of hydrolysis (injection every 30 min). Areas under the peaks of the starting materials and hydrolysis products were normalized according to the area of the peak of the internal standard.
The ed results are ted in figures 4 and 5. Noticeable hydrolysis was observed only for phenylmaleimide 1 (PhMal, kobs = 7x10‘ss'1). llshowed no detectable change in concentration. e 4: Stability of compounds of formula 11111 > Stabilit ofthe followin com ound in different conditions A 100 mM stock solution of the “addition product” NHBn was prepared in DMSO and stored at -20°C. 1 uL of the stock solution was added to 999 uL of working ons to give 100 uM final concentration of substrate. Aliquots were analyzed at 0, and 60 min. Areas under peak of starting material were normalized according to the area of the peak of the internal standard (benzamide). All measurements were carried at °C.
Table 3 below shows the conversion of “the addition t” in different media in one hour.
WO 20151001117 91 # Working solution Conversion of “addition product” in lh 100 mM PhSH in PBS(7.4):DMSO=80:20 Table 3 This ment clearly show that the addition product is stable and undergoes very little degradation in a wide range of conditions, in ular for pH ranging from 0 to In addition, hardly any thiol exchange is observed when the addition product is exposed for 1 hour to a medium sing an excess of another thiol, such as phenylthiol or glutathione. > Stabilit of a com ound of a III and com arison with maleimide The stability of compounds A (according to the invention) and B (reference compound) below was studied in different biological conditions.
Cell culture: WO 01117 92 Normal liver BNL CL.2 cells from mouse were grown in Dulbecco’s MEM medium with lg/l glucose (Eurobio, Les Ulis, France) supplemented with 10% fetal bovine serum (Perbio, res, France), 2 mM amine, 100 U/mL penicillin, 100 ug/mL streptomycin (Eurobio). Cells were maintained in a 5% C02 humidified atmosphere at 37°C.
Microscagy .' Twenty four hours prior to experiment, 2.5x104 BNL CL.2 cells were seeded per well in 8-well Lab-Tek II Chambered coverglass plates (ref 155409, Nunc, Naperville, IL, USA). The required amounts of probes A and B were diluted up to 300 pl in MEM complete medium to give final concentration of 1 uM and then added onto the cells. A 5 ug/ml of Hoechst ll8 solution was used as a nuclear marker. Cells were observed with a confocal Leica TSC SPE II microscope after washing with 10% FBS red phenol free Eagle’s MEM medium.
Cytometfl: The day before experiment, BNL CL.2 cells were seeded in 96-plates (Greiner Bio One, nhausen, Germany) at 2.0x104 cells/well in Dulbecco’s MEM complete .
Both probes (A and B) were prepared at 1 uM trations in Dulbecco’s MEM complete and added onto cells during different times (2, 6 and 24 hours). After washing with PBS (Eurobio), 5 min incubation with 40 ul trypsine, and addition of 160 ul of PBS EDTA 5 mM, cells were analyzed by flow cytometry on a PCA-96 Guava cytometer (Guava Technologies Merck ore, Billerica, MA, USA) with a green laser.
First, compound A was far more stable than compound B in human plasma (see fig 1).
Second, compound A was far more stable than compound B in o (see fig 2).
Example 4: Selectivity of compounds of the invention towards the thiol moiety Screening for selectivity was done on benzylamides of non-protected amino acids. 100 mM stock solutions of benzylamides of amino acids (in form of TFA salts) and electrophiles (phenylpropiolonitrile and phenylmaleimide) were prepared in DMSO and stored at -20°C. A 100 mM stock solution of benzamide (used as an internal standard) was prepared in led water and stocked at -20°C. Analyses of reaction mixtures W0 20151001117 93 were conducted with Shimadzu LC with SunFireTM C18 5 uM 0 mm column (Waters). HPLC parameters were as follows: flow rate 1 mL/min, gradient from 5 to 95% of mobile phase B from 0 to 20 min, followed by 5 min at 95% of mobile phase and post time of 5 min. Mobile phase A was 0.05% TFA in water (mQ) (V/V), and mobile phase B was acetonitrile (HPLC grade). Data were ed using Shimadzu analysis software. Signals were normalized according to the area of the peak of the internal standard (benzamide). Areas under the peaks of the amino acid benzylamides were used to calculate their sion during reaction. uL of the stock solution of amino acid benzamide and 2.5 uL of the stock solution of benzamide were added to a vial containing 977.5 uL of PBS (lx, pH 7.6). The solution was stirred and 10 uL of the stock solution of electrophile were added to give 1 mM final concentrations of reagents and 0.25 mM concentration of benzamide. Aliquots of the reaction mixture (50 uL) were analyzed by HPLC for 1 hour of hydrolysis (injections at 0, 30 and 60 min). Areas under the peaks of the starting materials and hydrolysis products were normalized ing to the area of peak of the internal standard. In case of phenylpropiolonitrile, none of amino acid models gave more than 1.6% conversion (see table 3 below). Conversely, when phenylmaleimide was tested, some amino acid benzylamides showed sions up to 8.5%. Masses of corresponding adducts were detected by mass spectrometry (ESI-LCMS) in some cases (shown in bold, Table 4).
Table 4 below presents the conversion of benzylamides in 1 hour in PBS (1X, pH 7.6) in ce of propiolonitrile (l).
Amino acid benzamide ‘WmmmmmcfliiiéigimdnwMMMMMMMMMM Amino acid benzamideml Conversion (‘ysNHBn {7a) 98% TerHBn (754‘) l 1.5% GlyNHBn. {7b} 0.39»; HisNHBn {711} 1 0.15% AlaNHBn (‘70) [IL 1% GluNHBu {7? 0.1% n (7.11 0.7% ”frpNHBu £15“? 1.3% SerNHBn (7e) 03% AJ'ENHBH (7k) 0.4% L MetNHBn or; J 0.1% J A§pNHBfl {'70 I 0.0020 Table 4 Table 5 below presents the conversion of benzylamides in 1 hour in PBS (1X, pH 7.6) in presence of phenylmaleimide. Bold values pond to those for which the mass of the corresponding adduct was detected by ESI-LCMS.
WO 20151001117 94 Amino acid benzamida Conversion ! Amino acid benzmnide [ Conversion zsNHBu {7a} 100 i 1* W331: r7 GWN‘EEIBI: (7b) ‘ HisN‘HBn (firm AlaNHBn {702 0.290" (WHNH {7%) W ‘ VaINHBn (7d) 0. 9% . TIpNHBu £31} 1.0% SerNHBn [7a) 2.8% if 7k) 2 .99»; 4 ArgNHBn MetNHBn (71') 1.9% AspNHBn (‘71) l. 3% Table 5 In conclusion, the selectivity of compounds of the invention towards the thiol moiety when compared to other moieties is clearly higher than the selectivity obtained for corresponding compounds, wherein the propiolonitrile moiety is replaced with a maleimide moiety.
Example 5: Toxicity tests Toxicity of the following “linker” compounds of formula (II) was studied by MTT assay on HaCaT cell lines: N N N N N N N N || || || II II || || || II II || || || || || || OMe NH2 N02 OMe NH2 NHAC 2 3 5 7 11 1O In Vitro cytotoxicity was measured using an MTT (3-(4,5-Dimethylthiazolyl)- 2,5-diphenyltetrazolium bromide) assay. The ments were med in 96—well plates with HaCaT cells grown to confluence in cell e media (RPMI 1640 media supplemented with 10% fetal calf serum and 1 mM Glutamin, 200 uL per well). Cells were ted with chemical reagent at different concentrations (100 uM to 0.78 uM by serial 1/2 dilutions) at 37°C for 24 hours. After tion, the supernatant was replaced with fresh culture media containing MTT (300 . After 2 hours of incubation at 37°C, the media was carefully removed and 100 uL ofDMSO were added WO 20151001117 95 to ve the formazan crystals generated by mitochondrial enzymes-induced reduction of the MTT. The absorbance was measured at 595 nm using a microplate reader (Biotek, y HT). The cell viabilities were sed as percent of untreated control cells.
The results of the MTT test are presented on figure 5, and clearly show that compounds of formula (II) are not toxic and may thus be used for instance for biological applications. e 6: ng of lysozyme with a nd of formula (11: Labeling of tryptic digest of lysozyme 1 nmol of me was solubilized in NH4HC03 (25 mM) and reduced with lmM of TCEP at 57°C during 1 hour. A solution of APN—TMPP (lmM in DMSO) was added to the protein at a molar ratio of 1:200. Then, d protein was subjected to proteolysis by porcine trypsin (Promega V5111). Sample was digested with 1:100 (w/w) trypsin in mM ammonium bicarbonate at 35 °C overnight. NanoLC-MS/MS analyses were performed to follow the reaction. The resulting peptide mixtures were analyzed by C18 reversed phase nanoHPLC on a nanoACQUITY Ultra-Performance-LC system (Waters, Milford, MA) coupled to a Q-TOF maXis (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with a nano—electrospray source. Chromatographic separation was performed on a nanoACQUITY Ultra-Performance-LC. The es were separated on an ACQUITY UPLC® BEH130 C18 column (Waters Corp), 75 um x 200 mm, 1.7 um particle size. The solvent system ted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Trapping was performed on a 20 x 0.18 mm, 5 um Symmetry C18 pre-column (Waters Corp.) during 3 minutes at 5 uL/min with 99% of solvent A and 1% of solvent B. Elution was performed at a flow rate of 300 nL/min, using a 1-50% gradient of solvent B for 30 minutes at 50°C followed by a fast rise at 80% (5 minutes) of solvent B. The complete system was fully controlled by Hystar 3.2 (Bruker ics). The Q—TOF instrument was operated with the following settings: source temperature was set to 200°C, drying gaz flow was 4 l/h, and the nano-electrospray voltage was 4kV. Mass ation of the TOP was achieved WO 20151001117 96 using ES-TOF Tuning Mix (Agilent Technologies) on the 50 to 2200 m/z range in positive mode. For tandem MS experiments, the system was operated with tic ing between MS and MS/MS modes both on m/z range [50-2200]. In MS the summation time was 0.2 s. In MS/MS summation time was weighted between 0.2 s and 1.4 s in fiinction of parent ion intensity. The 2 most abundant peptides (intensity old 400 au), preferably ions with two, three, four or five charges, were selected on each MS spectrum for further isolation and CID fragmentation with 2 energies set using collision energy profile. Fragmentation was performed using argon as the collision gas.
Tryptic peptides were manually sequenced (de novo) to confirm their sequence and locate the cysteine tagged by the AFN-TMPP probe. The peptides were identified using extracted ion chromatograms (EIC) based on monoisotopic mass of ated peptide sequences. tion of APN—TMPP chemoselectivity was carried by studying of its reaction with tryptic digest at 200:1 molar ratio of PP (luM) to protein (around 10:1 to cysteine moieties) at room temperature for one hour. Peptide mixtures obtained without and with chemical derivatization were analyzed by LC-MS/MS. All detectable cysteine- containing peptides reacted with a probe and were delayed while cysteine-free es were unaffected. The labeling efficiency was evaluated based on the ratio between ities of labeled and non-labeled peptides by LC-MS. More than 98% of the detected peptides were completely labeled. LC-MS results show y that cysteine- containing peptides have an sed retention time due to the addition of the hydrophobic TMPP group, whereas the retention time of all other peptides was unchanged (Table 6).
Table 6 below shows the results of LC-MS analyses of tryptic digest of me before and after reaction with APN—TMPP.
WO 20151001117 97 Elem“: tagging After g Pepwe sequencem r1112 (charge state} RT 1min} mix e stale) RT {min} ARR-11m} Number 91 tags “cswmmx” mam (+2) 13.9 545189 (+3.1 21.2 111.3 1 ”CELWMQXKZ" 426w 1+2) 116 551123 1+3} 2a? 1171 1 ”GrSLGvacAAK” 634131 (+2) 19.8 889.96H11) 2&3 as 1 Memo?" assess 1+2) 16.5 516122113} 25.? 9‘2 1 pcsmtssmmsmcm’“ 1mm {12; 23.7 919211 (+5} 30.3 as :1 ”Fasxmmnmn“ m“ 1331412} 1:15 1’1483 1+3) 137.5 a 0 ‘Werovmwmm 523127 1+2} am 52:12? 1+2) 1?.3 a a "N'Toesrommmssm 555123 1+2; 1331 535.23 (+2} 13.2 a 11 “*wsoommmwmwam 553.27 112) a u 559,27 1+2) ‘ 31149 ‘‘‘‘‘ “Maura V m {a} Cysteine residues are in hold“ Table 6 e 7: Conjugation of solubilizating APN reagents (49-54) with CD38 A275C mutant.
General scheme of the experiment (on the example of modification of CD38 mutant with 49) is illustrated on Figure 6.
To 300 ML of 75 solution (1 mg/mL) was added 6 ML of 50 mM solution of solubilizing APN reagent (49-54) in DMSO. In parallel, as a control, to 300 uL of CD38C275 was added 6 1.1L of DMSO. Both samples were incubated for 15 hours at 25 °C, then dialyzed 5 times (membrane cut off of 10k) to give final volume of 30 11L each (10 mg/mL). Size of aggregates was measured by DLS.
Exam le 8: Labelin of CD38-CD375 mutant and com arison with maleimide a) Stabilit of the com ound of the invention and of the corres ondin maleimide The compounds below were synthesized:.
WO 20151001117 98 Stability s proved that the compound according to the invention was stable for 24 hours in PBS (Phosphate Buffer Saline). atively, the ponding compound comprising a maleimide moiety was 70% degraded after one hour in PBS. b) Reaction with the CD38 mutant Both compounds were reacted with a 2 uM solution of the CD3 8-C375 mutant.
Gel electrophoresis after purification showed that a higher labeling rate could be obtained with the compounds according to the ion than with the ponding maleimide nd. Figure 7 presents the gel electrophoresis obtained with the compound of the invention and the ide, before and after purification.
Example 9: Conjugation of Trastuzumab and TAMRA using compound 18 General scheme of the experiment is illustrated on Figure 8.
To the solution of Trastuzumab (lOOuL, 10 mg/mL in 50mM borate buffer pH 8.5) was added 1.74 uL of the solution of 18 (10 mg/mL in DMSO). After incubation for 1h at °C was added 0.69 uL of TAMRA-SH (lOOmM in DMSO). The mixture was incubated at 25°C for 16h and the conjugate was purified by size exclusion chromatography.
The comparison experiment was carried out using 4-(N— maleimidomethyl)cyclohexanecarboxylic acid N—hydroxysuccinimide ester (SMCC) d of 18.
SDS-PAGE analysis of the obtained conjugates (Figure 9) showed that compound 18 allows for higher levels of conjugation comparing to SMCC.
Native ESI-MS analysis of the conjugate prepared using 18 (Figure 10, Figure 11) showed that in average one molecule ofTAMRA per antibody was conjugated.
Native ESI-MS analysis of the conjugate prepared using SMCC (Figure 12, Figure 13) showed a complex mixture ofundistinguishable species.
Experiment shows that the compound 18 allows for higher levels of conjugation and gives cleaner population of conjugates ing to generally applied SMCC.
WO 2015001117 99 Example 10: Direct conjugation of the compound 58 to partially reduced Trastuzumab.
General scheme of the experiment is illustrated on Figure 14.
To the solution of Trastuzumab , 10 mg/mL in 50mM PBS pH 7.4 with 10mM of EDTA) was added the solution of TCEP (10 mM in water, 1.1 or 2.2 eq.). The mixture was incubated at 37°C for 2h and then the solution of 58 (8.25 ML, 10 mM in DMSO) was added. The mixture was incubated at 25°C for 16h and the conjugate was purified by size exclusion chromatography.
GE is of the obtained conjugates (Figure 15) showed that compound 58 was covalently attached to the antibody. ESI-MS analysis showed that in e 4 molecules were conjugated per antibody using 2.2 eq. of TCEP. e 11: Rebridging of antibody fragments using compounds 33 and 34.
General scheme of the experiment is illustrated on Figure 16.
To the solution of Trastuzumab (lOOuL, 10 mg/mL in 50mM PBS pH 7.4 with 10mM of EDTA) was added the solution of TCEP (10 mM in water, 5 eq.). The mixture was incubated at 37°C for 2h and then the solution of 33 or 34 (10 mM in DMSO, 15 eq.) was added. The resulting solution was incubated for 16 h at 25°C and then analyzed by SDS-PAGE in reducing conditions.
SDS-PAGE analysis showed that antibody fragments were successfully bridged by compounds 33 and 34 (Figure 17).

Claims (4)

1. Process for bioconjugation of a protein sing at least one cysteine residue having a thiol moiety with an antibody, a drug or a fluorescent probe, comprising 5 contacting a protein comprising at least one cysteine residue having a thiol moiety with a compound of formula (I): (I) , wherein each of R1 to R5 is independently selected in the group consisting of: - hydrogen atoms, 10 - alkyl, alkene or alkyne groups, ally interrupted by at least one heteroatom selected among O, N and S, - aryl groups, - alkoxy groups, - halogen atoms, 15 - -NRR’ , - -ONH2 group, - -NH-NH2 group, - -NO2 group, - -N3 group, 20 - -N2+ group, - maleimide group, - -C(=O)OR groups, - -C(=O)R groups, - -OH group, 25 - -B(OR’’)2 group, - phosphine or phosphonium groups, - -N=C=O or -N=C=S group, - -SO2Cl group, - a -O-C(=O)-C(N2)-CF3 group or a -C(=O)-C(N2)-CF3 group, 30 - activated esters, such as -NHS, perfluorinated esters and acylureas, - a -C≡C-C≡N group, and - alkyl groups tuted by at least one of the previously listed groups, wherein R and R’ are independently hydrogen atoms, alkyl, alkene, alkyne or aryl groups and R’’ is a hydrogen atom or an alkyl group, and 5 wherein at least one of R1 to R5 comprises a tag moiety which is optionally bonded to the phenyl ring through a linker group and which is selected from an antibody; a drug; or a fluorescent probe.
2. A compound of the formula (I): 10 (I) , wherein R1 to R5 are as d in claim 1, provided that the compound is not [3-(3-formyl-phenylethynyl)-phenyl]-propyne nitrile
3. A compound selected from the group consisting of: 103 103 \ / o ”N O INI INI 26 27 l \ N | \ N |+ \ —FL+— \ l ‘ N\ Hm) _N+_ N H 0 o /\ 0 O 0 F F 0 / GAO F F / F F \ I / FmocHN F F \ I F F SO3Na \ F F F O OH 32 SO3Na 33 36 N N H H _N/ N C L £1 \9 O O O 18F CF218F 125' L \\ 37 38 39 43 44 \\N HN O 9 . 5
4. A nd of formula (III): (III) , wherein R1 to R5 are as described in claim 1, and wherein R6-SH is a protein comprising at least one cysteine residue having a thiol moiety. W0 20151001117
NZ715706A 2013-07-04 2014-07-04 3-aryl propiolonitrile compounds for thiol labeling NZ715706B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP13305950.1A EP2821791A1 (en) 2013-07-04 2013-07-04 3-aryl propiolonitrile compounds for thiol labeling
EP13305950.1 2013-07-04
PCT/EP2014/064387 WO2015001117A1 (en) 2013-07-04 2014-07-04 3-aryl propiolonitrile compounds for thiol labeling

Publications (2)

Publication Number Publication Date
NZ715706A true NZ715706A (en) 2020-12-18
NZ715706B2 NZ715706B2 (en) 2021-03-19

Family

ID=

Also Published As

Publication number Publication date
US9663454B2 (en) 2017-05-30
CA2915733A1 (en) 2015-01-08
WO2015001117A1 (en) 2015-01-08
CN108069876A (en) 2018-05-25
AU2014286066A1 (en) 2016-01-28
EP3017307A1 (en) 2016-05-11
EP3017307B1 (en) 2018-11-21
JP6578275B2 (en) 2019-09-18
ES2712178T3 (en) 2019-05-09
JP2016536292A (en) 2016-11-24
US20170210700A1 (en) 2017-07-27
EP2821791A1 (en) 2015-01-07
CA2915733C (en) 2021-07-20
US20160145199A1 (en) 2016-05-26
AU2014286066B2 (en) 2020-04-16
US10131626B2 (en) 2018-11-20
CN105474015A (en) 2016-04-06
CN105474015B (en) 2018-01-26

Similar Documents

Publication Publication Date Title
AU2014286066B2 (en) 3-aryl propiolonitrile compounds for thiol labeling
US6207831B1 (en) Fluorescent dyes (AIDA) for solid phase and solution phase screening
CS273617B2 (en) Method of resorufine&#39;s derivatives production
JP2023520940A (en) Targeted Protease Degradation (TED) Platform
Chen et al. An efficient and versatile approach for the preparation of a rhodamine B ester bioprobe library
Caron et al. Dramatic increase of quench efficiency in “spacerless” dimaleimide fluorogens
EP3735987A1 (en) Amanitin antibody conjugate
US6403625B1 (en) Fluorescent labeling reagents
Wängler et al. Improved syntheses and applicability of different DOTA building blocks for multiply derivatized scaffolds
Nagy et al. An acrylated isocyanonaphthalene based solvatochromic click reagent: Optical and biolabeling properties and quantum chemical modeling
JP7231621B2 (en) Novel water-soluble mono- and bi-branched complexing agents and corresponding lanthanide complexes
Banerjee et al. Design and synthesis of site directed maleimide bifunctional chelators for technetium and rhenium
Clavé et al. A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates
NZ715706B2 (en) 3-aryl propiolonitrile compounds for thiol labeling
US20100221749A1 (en) Three-functional pseudo-peptidic reagent, and uses and applications thereof
WO2021177050A1 (en) Activatable raman probe
CN114409563B (en) Linker for protein labeling and application thereof in biological medicine
WO2019168164A1 (en) Molecule for protein and/or peptide design
JP4929452B2 (en) New coumarin derivatives
JP6670502B2 (en) Development of ligand screening system for neurotransmitter receptor
Balázsi et al. Synthesis of 3, 4-Disubstituted Pyrroline Nitroxides Containing Diphenylphosphane or Diphenylphosphane Oxide Substituents
Folliet Towards the development of a visible light-enabled histidine-selective protein modification strategy using diazo sulfonium-based probes
JP2019500354A (en) Water-soluble triazapyridinophan complexing agents and corresponding fluorescent lanthanide complexes
Selaya Investigations into the fluorescent covalent labeling of biomolecules utilizing rhodamine dyes, electrophilic leaving groups and mRNA display
WO2022038359A1 (en) Cross-linking method and applications in bioconjugation

Legal Events

Date Code Title Description
PSEA Patent sealed
RENW Renewal (renewal fees accepted)

Free format text: PATENT RENEWED FOR 1 YEAR UNTIL 04 JUL 2022 BY IPAN GMBH

Effective date: 20210618

RENW Renewal (renewal fees accepted)

Free format text: PATENT RENEWED FOR 1 YEAR UNTIL 04 JUL 2023 BY IPAN GMBH

Effective date: 20220613

RENW Renewal (renewal fees accepted)

Free format text: PATENT RENEWED FOR 1 YEAR UNTIL 04 JUL 2024 BY IPAN GMBH

Effective date: 20230613